Anti-talen antibodies and uses thereof

ABSTRACT

The present disclosure provides, among other things, antibodies for detecting TALENs and/or FokI nucleases in a sample and methods of using the same.

CROSS REFERENCE

This application is a divisional of U.S. application Ser. No.16/900,602, filed Jun. 12, 2020, which claims the benefit of priority toU.S. Provisional Application No. 62/861,214, filed on Jun. 13, 2019, thecontent of both of which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

This application is being filed electronically via Patent Center andincludes an electronically submitted sequence listing in .xml format.The .xml file contains a sequence listing entitled “AT-021_03_SL.xml”created on Jul. 17, 2023, and having a size of 38,698 bytes. Thesequence listing contained in this .xml file is part of thespecification and is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to antigen binding molecules, including,but not limited to, antibodies, which specifically bind gene editingproteins (e.g., TALEN proteins comprising a FokI, also referred to asFok-1 or Fok1, catalytic domain) and polynucleotides encoding the same,and methods of use.

BACKGROUND

Genome editing tools such as Transcription activator-like effectornucleases (TALEN®), Zinc-finger nucleases (ZFNs), and RNA-guided FokINucleases (RFNs) are powerful tools that enable the precise revision,insertion or deletion of genes. TALENs, ZFNs, and RFNs can bind to andcleave at targeted sites in a genome. The resulting cleaved DNA isrepaired via the endogenous cell DNA repair mechanism of non-homologousend joining (NHEJ), or via homology-directed repair (HDR) when a repairtemplate is provided. TALENs use a TAL effector DNA-binding domain fusedto a DNA cleavage domain (e.g., FokI). ZFNs are composed of arestriction endonuclease cleavage or catalytic domain, such as FokI,linked to engineered Cys₂His₂ zinc-finger, DNA-binding polypeptides.RFNs are composed of a FokI nuclease fused to catalytically inactiveform of Cas9. For efficient genome editing in mammals (e.g., humans,dogs, cattle, non-human primates, etc.), it is envisioned that TALENs,ZFNs and RFNs will be expressed at high level. Due to the risk ofsustained nuclease activity and off-target effects, as well as potentialimmunogenicity, there is a need in the art for antibodies that recognizegenome editing tools (e.g., anti-TALEN and/or anti-FokI antibodies)capable of binding nuclease proteins in order to evaluate the safety andefficacy of various gene editing therapies.

SUMMARY OF THE INVENTION

The present disclosure provides, among other things, antibodies for thedetection of FokI catalytic domains and full-length TALEN proteins in asample. As described herein, including in the Examples below, thepresent disclosure provides anti-TALEN antibodies which specificallybind TALEN and FokI proteins. In some embodiments anti-TALEN antibodiesof the present disclosure can be used to evaluate gene editing therapiesand processes.

In one aspect, the instant disclosure provides an anti-TALEN antibodycomprising a variable heavy chain amino acid sequence that is at least85% identical to SEQ ID NO: 3 and a variable light chain amino acidsequence that is at least 85% identical to SEQ ID NO: 7. In oneembodiment the anti-TALEN antibody is humanized. In various embodiments,the antibody is selected from the group consisting of an antibody, anscFv, a Fab, a Fab′, a Fv, a F(ab′)₂, a dAb, a human antibody, ahumanized antibody, a chimeric antibody, a monoclonal antibody, apolyclonal antibody, a recombinant antibody, an IgE antibody, an IgDantibody, an IgM antibody, an IgG1 antibody, an IgG1 antibody having atleast one mutation in the hinge region, an IgG2 antibody an IgG2antibody having at least one mutation in the hinge region, an IgG3antibody, an IgG3 antibody having at least one mutation in the hingeregion, an IgG4 antibody, an IgG4 antibody having at least one mutationin the hinge region, an antibody comprising at least one non-naturallyoccurring amino acid, and any combination thereof.

In specific embodiments, the antibody comprises a variable heavy chain(HC) selected from the group consisting of SEQ ID NOs: 3-6, and infurther embodiments the anti-TALEN antibody comprises a heavy chain CDR1selected from the group consisting of SEQ ID NOs: 12-14, and/or a heavychain CDR2 selected from the group consisting of SEQ ID NOs: 15-20,and/or a heavy chain CDR3 comprising SEQ ID NO: 21. In specificembodiments, an anti-TALEN antibody comprises a heavy chain CDR1, aheavy chain CDR2, and a heavy chain CDR3, each CDR comprising an aminoacid sequence shown in Table 1c. Also provided is an anti-TALEN antibodycomprising a VH amino acid sequence that is at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or about 100% identical to a VH of an antigenbinding molecule provided herein.

In other embodiments an anti-TALEN antibody comprises a light chainvariable region (VL) sequence selected from the group consisting of SEQID NOs: 7-11. In specific embodiments, an anti-TALEN antibody comprisesa light chain CDR1 comprising SEQ ID NO: 22, and/or a light chain CDR2selected from the group consisting of SEQ ID NO: 23-25, and/or a lightchain CDR3 selected from the group consisting of SEQ ID NOs: 26-28.Further provided is an anti-TALEN antibody wherein the light chaincomprises a light chain CDR1, a light chain CDR2 and a light chain CDR3,each CDR comprising an amino acid sequence in Table ld. Also provided isan anti-TALEN antibody, comprising a VL amino acid sequence that is atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or about 100% identical toa VL of an antigen binding molecule provided herein.

In another embodiment, provided herein is an anti-TALEN antibody,comprising (a) a VH CDR1 comprising the amino acid sequence of SEQ IDNO:12; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 21;(d)a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22; (e) aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) aVL CDR3 comprising the amino acid sequence of SEQ ID NO: 26 and in afurther embodiment, an anti-TALEN antibody comprises (a) a VH comprisingthe amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising theamino acid sequence of SEQ ID NO: 7.

In another embodiment provided herein is an anti-TALEN antibody,comprising (a) a VH CDR1 comprising the amino acid sequence of SEQ IDNO: 12; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:16; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 21;(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22; (e) aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) aVL CDR3 comprising the amino acid sequence of SEQ ID NO: 27, and in afurther embodiment an anti-TALEN antibody comprises (a) a VH comprisingthe amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising theamino acid sequence of SEQ ID NO: 8.

In a further embodiment, provided herein is an anti-TALEN antibodycomprising (a) a VH CDR1 comprising the amino acid sequence of SEQ IDNO: 13; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:16; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 21;(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22; (e) aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) aVL CDR3 comprising the amino acid sequence of SEQ ID NO: 26, and in afurther embodiment the anti-TALEN antibody comprises (a) a VH comprisingthe amino acid sequence of SEQ ID NO: 4; and (b) a VL comprising theamino acid sequence of SEQ ID NO: 9.

In still a further embodiment, provided herein is an anti-TALEN antibody

comprising (a) a VH CDR1 comprising the amino acid sequence of SEQ IDNO: 12; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 21;(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22; (e) aVL CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and (f) aVL CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and in afurther embodiment the anti-TALEN antibody comprises (a) a VH comprisingthe amino acid sequence of SEQ ID NO: 5; and (b) a VL comprising theamino acid sequence of SEQ ID NO: 10.

Also provided is an anti-TALEN antibody, comprising (a) a VH CDR1comprising the amino acid sequence of SEQ ID NO: 12; (b) a VH CDR2comprising the amino acid sequence of SEQ ID NO: 17; (c) a VH CDR3comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 24; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO: 27, and in a furtherembodiment the anti-TALEN antibody of claim 23, comprising (a) a VHcomprising the amino acid sequence of SEQ ID NO: 6; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 11.

In various embodiments the anti-TALEN antibody further comprises adetectable label, and in some embodiments the detectable label isselected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten. When the detectable label is afluorescent label, the fluorescent label can be selected from the groupconsisting of an Atto dye, an Alexafluor dye, quantum dots,Hydroxycoumarin, Aminocouramin, Methoxycourmarin, Cascade Blue, PacificBlue, Pacific Orange, Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX,Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC,X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC),APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP,Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, KusabiraOrange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP,DsRed monomer, DsRed2 (“RFP”), mStrawberry, TurboFP602, AsRed2, mRFP1,J-Red, R-phycoerythrin (RPE), B-phycoeryhring (BPE), mCherry, HcRed1,Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635),TurboFP635, mPlum, and mRaspberry. In specific embodiments thefluorescent label is R-Phycoerythrin (PE) or Allophycocyanin (APC)

Also provided is a composition comprising an anti-TALEN antibodyprovided herein and an acceptable carrier or vehicle, e.g., apharmaceutically acceptable carrier or vehicle.

Polynucleotides encoding the heavy chain of an anti-TALEN antibodydisclosed herein are also provided. Polynucleotide encoding the lightchain of an anti-TALEN antibody disclosed herein are also provided.Vectors comprising one or both of the heavy and light chain encodingpolynucleotides are also provided. Further, a cell comprising one orboth of the vectors is provided, and the cell can comprise a cellselected from the group consisting of a CHO cell, a Sp2/0 cell, a rabbitcell and an E. coli cell. A method of making an anti-TALEN antibodydisclosed herein is provided and comprises incubating a cell comprisingone or both of the vectors described above under suitable conditions.

In yet a further embodiment a method of determining the presence or

absence of a TALEN protein in a sample is provided and in one embodimentcomprises contacting the sample with an anti-TALEN antibody conjugatedto a detectable label and determining the presence or absence of theTALEN protein in the sample; in some embodiments the anti-TALEN antibodycomprises an antibody provided herein, or a humanized form thereof.Additionally, the sample can be a tissue sample, a blood sample, aformalin-fixed sample, a tissue grown ex vivo or cell culture media.

Various aspects of the present disclosure are described in detail in thefollowing sections. The use of sections is not meant to limit thepresent disclosure. Each section can apply to any aspect of the presentdisclosure. In this application, the use of “or” means “and/or” unlessstated otherwise. All references cited herein are incorporated byreference in their entirety. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the presentdisclosure belongs.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings provided in the present disclosure are for illustrationpurposes only and not for limitation.

FIG. 1 depicts a Western blot analysis of hybridoma supernatants fromfusion #2299 (Numbers 13-16 noted in the figure) and #2296 (Numbers 3,6, 7, 8, and 11 noted in the figure).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in the arts of genetic engineering, cell culture,molecular genetics, nucleic acid chemistry and biochemistry).

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout the presentdisclosure.

Affinity: As is known in the art, “affinity” refers to a measure of thetightness that a particular ligand binds to its partner. Affinities canbe measured in different ways. In some embodiments, affinity is measuredby a quantitative assay. In some embodiments, binding partnerconcentration and/or ligand concentration can be varied. In some suchembodiments, affinity can be compared to a reference under comparableconditions (e.g., concentrations).

Amino acid: in its broadest sense, refers to any compound and/orsubstance that can be incorporated into a polypeptide chain, e.g.,through formation of one or more peptide bonds. In some embodiments, anamino acid has the general structure H₂N—C(H)(R)—COOH. In someembodiments, an amino acid is a naturally-occurring amino acid. In someembodiments, an amino acid is a synthetic amino acid; in someembodiments, an amino acid is a D-amino acid; in some embodiments, anamino acid is an L-amino acid. The term “standard amino acid” refers toany of the twenty standard L-amino acids commonly found in naturallyoccurring peptides. The term “nonstandard amino acid” refers to anyamino acid, other than the standard amino acids, regardless of whetherit is prepared synthetically or obtained from a natural source. In someembodiments, an amino acid, including a carboxy- and/or amino-terminalamino acid in a polypeptide, can contain a structural modification ascompared with the general structure above. For example, in someembodiments, an amino acid can be modified by methylation, amidation,acetylation, and/or substitution as compared with the general structure.As will be clear from context, in some embodiments, the term “aminoacid” is used to refer to a free amino acid; in some embodiments it isused to refer to an amino acid residue of a polypeptide.

Antibody: As used herein, the term “antibody” refers to a species ofantigen binding proteins comprising a polypeptide that includescanonical immunoglobulin sequence elements sufficient to confer specificbinding to a particular target antigen. As is known in the art, intactantibodies as produced in nature are approximately 150 kD tetramericagents comprised of two identical heavy chain polypeptides (about 50 kDeach) and two identical light chain polypeptides (about 25 kD each) thatassociate with each other into what is commonly referred to as a“Y-shaped” structure. Each heavy chain is comprised of at least fourdomains (each about 110 amino acids long), an amino-terminal variable(VH) domain (located at the tips of the Y structure), followed by threeconstant domains: CH1, CH2, and the carboxy-terminal CH3 (located at thebase of the Y′s stem). A short region, commonly referred to as the“switch”, connects the heavy chain variable and constant regions. The“hinge” connects CH2 and CH3 domains to the rest of the antibody.

Two disulfide bonds in this hinge region connect the two heavy chainpolypeptides to one another in an intact antibody. Each light chain iscomprised of two domains—an amino-terminal variable (VL) domain,followed by a carboxy-terminal constant (CL) domain, separated from oneanother by another “switch”. Intact antibody tetramers are comprised oftwo heavy chain-light chain dimers in which the heavy and light chainsare linked to one another by a single disulfide bond; two otherdisulfide bonds connect the heavy chain hinge regions to one another, sothat the dimers are connected to one another and the tetramer is formed.Naturally-produced antibodies are also glycosylated, typically on theCH2 domain. Each domain in a natural antibody has a structurecharacterized by an “immunoglobulin fold” formed from two beta sheets(e.g., 3-, 4-, or 5-stranded sheets) packed against each other in acompressed antiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1,FR2, FR3, and FR4). When natural antibodies fold, the FR regions formthe beta sheets that provide the structural framework for the domains,and the CDR loop regions from both the heavy and light chains arebrought together in three-dimensional space so that they create a singlehypervariable antigen binding site located at the tip of the Ystructure. The Fc region of naturally-occurring antibodies binds toelements of the complement system, and also to receptors on effectorcells, including for example effector cells that mediate cytotoxicity.In some embodiments, an antibody is polyclonal; in some embodiments, anantibody is monoclonal.

Binding: It will be understood that the term “binding”, as used herein,typically refers to a non-covalent association between or among two ormore entities. “Direct” binding involves physical contact betweenentities or moieties; “indirect” binding involves physical interactionby way of physical contact with one or more intermediate entities.

Chromosome: As used herein, the term “chromosome” refers to a linearmolecule of DNA with associated proteins in the nucleus of eukaryoticcells that carries the genes and functions in the transmission ofhereditary information.

Corresponding to: as used herein designates the position/identity of anamino acid residue in a polypeptide of interest. Those of ordinary skillwill appreciate that, for purposes of simplicity, residues in apolypeptide are often designated using a canonical numbering systembased on a reference related polypeptide, so that an amino acid“corresponding to” a residue at position 190, for example, need notactually be the 190^(th) amino acid in a particular amino acid chain butrather corresponds to the residue found at 190 in the referencepolypeptide; those of ordinary skill in the art readily appreciate howto identify “corresponding” amino acids.

Detection moiety: The term “detection moiety” as used herein refers toany element, molecule, functional group, compound, fragment or moietythat is detectable. In some embodiments, a detection moiety is providedor utilized alone. In some embodiments, a detection moiety is providedand/or utilized in association with (e.g., joined to) another agent.Examples of detection moieties include, but are not limited to: variousligands, radionuclides (e.g., ³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵I, ¹²⁵I,¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y, ^(99m)Tc, ¹⁷⁷Lu, ⁸⁹Zr etc.), fluorescentdyes (such as, for example fluorescein dyes, acridine dyes, SYBR dyes,rhodamine dyes, oxazine dyes, etc.), chemiluminescent agents (such as,for example, acridinum esters, stabilized dioxetanes, and the like),electrochemiluminescent agents (such as, for example, Sulfo Tags),bioluminescent agents (such as, for example, luciferin), spectrallyresolvable inorganic fluorescent semiconductors nanocrystals (i.e.,quantum dots), metal nanoparticles (e.g., gold, silver, copper,platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes (suchas, for example, horseradish peroxidase, alkaline phosphatase, etc.),colorimetric labels (such as, for example, dyes, colloidal gold, and thelike), biotin, digoxigenin, haptens, and proteins for which antisera ormonoclonal antibodies are available.

Engineered: In general, the term “engineered” refers to the aspect ofhaving been manipulated by the hand of man. For example, apolynucleotide is considered to be “engineered” when two or moresequences that are not linked together in that order in nature aremanipulated by the hand of man to be directly linked to one another inthe engineered polynucleotide. For example, in some embodiments of thepresent disclosure, an engineered polynucleotide can comprise aregulatory sequence that is found in nature in operative associationwith a first coding sequence but not in operative association with asecond coding sequence, is linked by the hand of man so that it isoperatively associated with the second coding sequence. Comparably, acell or organism is considered to be “engineered” if it has beenmanipulated so that its genetic information is altered (e.g., newgenetic material not previously present has been introduced, for exampleby transformation, mating, somatic hybridization, transfection,transduction, or other mechanism, or previously present genetic materialis altered or removed, for example by substitution or by a deletionmutation, or by mating protocols). As is common practice and isunderstood by those in the art, progeny of an engineered polynucleotideor cell are typically still referred to as “engineered” even though theactual manipulation was performed on a prior entity.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; and/or (4) post-translational modification of a polypeptide orprotein.

FokI: As used herein, the term “FokI” (alternatively referred to as Fok1or Fok-1) refers to a bacterial type IIS restriction endonuclease thatis naturally found in Flavobacterium okeanokoites. Natural FokIcomprises of an N-terminal DNA-binding domain and a non-specific DNAcleavage domain at the C-terminus. The FokI DNA cleavage domain canfunction independently of the DNA binding domain. As used herein, FokIrefers to a protein or a nucleic acid encoding a protein that comprisesthe DNA cleavage domain (i.e., the nuclease domain) of FokI. FokI canrefer to the entire FokI protein (SEQ ID NO: 2) or just the catalyticnuclease domain (SEQ ID NO: 1). The term “FokI” refers to all fragmentsand variants of FokI including mutants, derivatives, analogs,truncations, fusion constructs, and multimers of FokI, including FokIdimers. In some embodiments, FokI is recombinantly produced. In someembodiments, a FokI polypeptide is conjugated to or tagged with anadditional moiety, such as a binding moiety and/or a detection moiety.As will be clear from context, in some embodiments, the term “FokI” isused to refer to the FokI polypeptide or fragments or variants thereofin some embodiments “FokI” is used to refer to nucleic acids encoding aFokI polypeptide and fragments and variants thereof.

Fragment: A “fragment” of a biological molecule as described herein hasa structure that includes a discrete portion of the whole but lacks oneor more moieties found in the whole (e.g., a polypeptide lacking one ormore amino acids). In some embodiments, a fragment consists of such adiscrete portion. In some embodiments, a fragment consists of orcomprises a characteristic structural element or moiety found in thewhole (e.g. a polypeptide domain). In some embodiments, the biologicalmolecule is a polymer (e.g. a nucleic acid polymer or a polypeptide). Insome embodiments, a polymer fragment comprises or consists of at least3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g.,residues) as found in the whole polymer. In some embodiments, a polymerfragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%,30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) foundin the whole polymer. In some embodiments, the whole biological moleculecan be referred to as the “parent” of the whole.

Fusion: A “fusion molecule” is a molecule in which two or more subunitmolecules are linked, preferably covalently. The subunit molecules canbe the same chemical type of molecule or can be different chemical typesof molecules. Examples fusions of the same chemical type of moleculeinclude, but are not limited to, fusion proteins (for example, a fusionbetween a Zinc Finger Protein or TALEN DNA-binding domain to a FokIcleavage domain) and fusion nucleic acids (for example, a nucleic acidencoding the fusion protein described supra). Examples fusions ofdifferent chemical types of molecule include, but are not limited to,nucleic acid-polypeptide fusions (for example, a fusion between an RNAmolecule and a FokI cleavage domain).

Gene: As used herein, the term “gene” refers to a DNA sequence thatcodes for a product (e.g., an RNA product and/or a polypeptide product).In some embodiments, a gene includes coding sequence (i.e., sequencethat encodes a particular product); in some embodiments, a gene includesnon-coding sequence. In some particular embodiments, a gene can includeboth coding (e.g., exonic) and non-coding (e.g., intronic) sequence. Insome embodiments, a gene can include one or more regulatory elementsthat, for example, can control or impact one or more aspects of geneexpression (e.g., cell-type-specific expression, inducible expression,etc).

Gene or Cell therapy: As used herein, the term “gene or cell therapy”refers to insertion or deletion of specific genomic DNA sequences totreat or prevent a disorder or condition for which such therapy issought. In some embodiments, the insertion or deletion of genomic DNAsequences occurs in specific cells (i.e. target cells). Target cells canbe from a mammal and/or can be cells in a mammalian subject. Mammalsinclude but are not limited to humans, dogs, cats, cows, sheep, pigs,llamas, non-human primates, etc. In some embodiments, heterologous DNAis transferred to target cells. The heterologous DNA can be introducedinto the selected target cells in a manner such that the heterologousDNA is expressed, and a therapeutic product encoded thereby is produced.Additionally, or alternatively, the heterologous DNA can in some mannermediate expression of DNA that encodes the therapeutic product, or itcan encode a product, such as a peptide or RNA that in some mannermediates, directly or indirectly, expression of a therapeutic product.Genetic or cell therapy can also be used to deliver nucleic acidencoding a gene product that replaces a defective gene or supplements agene product produced by the mammal or the cell in which it isintroduced. The heterologous DNA encoding the therapeutic product can bemodified prior to introduction into the cells of the afflicted subjectin order to enhance or otherwise alter the product or expressionthereof. Genetic or cell therapy can also involve delivery of aninhibitor or repressor or other modulator of gene expression. Genetherapy can include in vivo or ex vivo techniques. In some embodiments,gene or cell therapy includes administering nucleic acids encodingTranscription activator-like effector nucleases (TALENs), Zinc-FingerNucleases (ZFNs) or RNA-guided FokI nucleases (RFNs). In someembodiments, viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding TALENs, ZFNs or RFNs into mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding TALENs, ZFNs or RFNs to cells in vitro. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as poloxamers or liposomes.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see, e.g., Anderson, Science256:808-813 (1992); Miller, Nature 357:455-460 (1992); Feuerbach et al.,Kidney International 49:1791-1794 (1996); Urnov et al., Nature ReviewsGenetics 11, 636-646 (2010); and Collins et al., Proceedings BiologicialSciences/The Royal Society, 282(1821):pii 20143003 (2015).

Genome: As used herein, the term “genome” refers to the total geneticinformation carried by an individual organism or cell, represented bythe complete DNA sequences of its chromosomes.

High affinity binding: The term “high affinity binding”, as used hereinrefers to a high degree of tightness with which a particular ligandbinds to its partner. Affinities can be measured by any availablemethod, including those known in the art. In some embodiments, bindingis considered to be high affinity if the K_(d) is about 500 pM or less(e.g., below about 400 pM, about 300 pM, about 200 pM, about 100 pM,about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about40 pM, about 30 pM, about pM, about 10 pM, about 5 pM, about 4 pM, about3 pM, about 2 pM, etc.) in binding assays. In some embodiments, bindingis considered to be high affinity if the affinity is stronger (e.g., theK_(d) is lower) for a polypeptide of interest than for a selectedreference polypeptide. In some embodiments, binding is considered to behigh affinity if the ratio of the K_(d) for a polypeptide of interest tothe K_(d) for a selected reference polypeptide is 1:1 or less (e.g.,0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1. 0.4:1, 0.3:1, 0.2:1, 0.1:1, 0.05:1,0.01:1, or less). In some embodiments, binding is considered to be highaffinity if the K_(d) for a polypeptide of interest is about 100% orless (e.g., about 99%, about 98%, about 97%, about 96%, about 95%, about90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%,about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%,about 2%, about 1% or less) of the K_(d) for a selected referencepolypeptide.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments,polymeric molecules are considered to be “homologous” to one another iftheir sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar.

In vitro: The term “in vitro” as used herein refers to events that occurin an artificial environment, e.g., in a test tube or reaction vessel,in cell culture, etc., rather than within a multi-cellular organism.

In vivo: as used herein refers to events that occur within amulti-cellular organism, such as a human and a non-human animal. In thecontext of cell-based systems, the term can be used to refer to eventsthat occur within a living cell (as opposed to, for example, in vitrosystems).

Isolated: as used herein, refers to a substance and/or entity that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities canbe separated from about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% of the other components with which they wereinitially associated. In some embodiments, isolated agents are about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. In some embodiments, as will beunderstood by those skilled in the art, a substance can still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. To give but one example, in someembodiments, a biological polymer such as a polypeptide orpolynucleotide that occurs in nature is considered to be “isolated”when, a) by virtue of its origin or source of derivation is notassociated with some or all of the components that accompany it in itsnative state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; c) is expressed by or is otherwise in associationwith components from a cell or other expression system that is not ofthe species that produces it in nature. Thus, for instance, in someembodiments, a polypeptide that is chemically synthesized or issynthesized in a cellular system different from that which produces itin nature is considered to be an “isolated” polypeptide. Alternativelyor additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques can be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

Linker: as used herein, is used to refer to that portion of amulti-element polypeptide that connects different elements to oneanother. For example, those of ordinary skill in the art appreciate thata polypeptide whose structure includes two or more functional ororganizational domains often includes a stretch of amino acids betweensuch domains that links them to one another. In some embodiments, apolypeptide comprising a linker element has an overall structure of thegeneral form S1-L-S2, wherein S1 and S2 can be the same or different andrepresent two domains associated with one another by the linker. In someembodiments, a linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more aminoacids in length. In some embodiments, a linker is characterized in thatit tends not to adopt a rigid three-dimensional structure, but ratherprovides flexibility to the polypeptide. A variety of different linkerelements that can appropriately be used when engineering polypeptides(e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., etal. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., etal. (1994) Structure 2: 1 121-1123).

Nucleic acid: as used herein, in its broadest sense, the term “nucleicacid” refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments, anucleic acid is a compound and/or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage.As will be clear from context, in some embodiments, “nucleic acid”refers to individual nucleic acid residues (e.g., nucleotides and/ornucleosides); in some embodiments, “nucleic acid” refers to anoligonucleotide chain comprising individual nucleic acid residues. Insome embodiments, a “nucleic acid” is or comprises RNA; in someembodiments, a “nucleic acid” is or comprises DNA. In some embodiments,a nucleic acid is, comprises, or consists of one or more natural nucleicacid residues. In some embodiments, a nucleic acid is, comprises, orconsists of one or more nucleic acid analogs. In some embodiments, anucleic acid has a nucleotide sequence that encodes a functional geneproduct such as an RNA or protein. In some embodiments, a nucleic acidincludes one or more introns. In some embodiments, nucleic acids areprepared by one or more of isolation from a natural source, enzymaticsynthesis by polymerization based on a complementary template (in vivoor in vitro), reproduction in a recombinant cell or system, and chemicalsynthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residueslong. In some embodiments, a nucleic acid is single stranded; in someembodiments, a nucleic acid is double stranded. In some embodiments anucleic acid has a nucleotide sequence comprising at least one elementthat encodes, or is the complement of a sequence that encodes, apolypeptide. In some embodiments, a nucleic acid has enzymatic activity.

Physiological conditions: as used herein, has its art-understood meaningreferencing conditions under which cells or organisms live and/orreproduce. In some embodiments, the term refers to conditions of theexternal or internal milieu that can occur in nature for an organism orcell system. In some embodiments, physiological conditions are thoseconditions present within the body of a human or non-human animal,especially those conditions present at and/or within a surgical site.Physiological conditions typically include, e.g., a temperature range ofabout 20-about 40° C., atmospheric pressure of about 1, pH of about6-about8, glucose concentration of about 1-about 20 mM, oxygenconcentration at atmospheric levels, and gravity as it is encountered onearth. In some embodiments, conditions in a laboratory are manipulatedand/or maintained at physiologic conditions. In some embodiments,physiological conditions are encountered in an organism.

Polypeptide: The term “polypeptide”, as used herein, generally has itsart-recognized meaning of a polymer of at least three amino acids. Thoseof ordinary skill in the art will appreciate that the term “polypeptide”is intended to be sufficiently general as to encompass not onlypolypeptides having a complete sequence recited herein, but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those of ordinary skill in the art understandthat protein sequences generally tolerate some substitution withoutdestroying activity. Thus, any polypeptide that retains activity andshares at least about 30-40% overall sequence identity, often greaterthan about 50%, 60%, 70%, or 80%, and further usually including at leastone region of much higher identity, often greater than 90% or even 95%,96%, 97%, 98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term “polypeptide” as used herein. Polypeptides can containL-amino acids, D-amino acids, or both and can contain any of a varietyof amino acid modifications or analogs known in the art. Usefulmodifications include, e.g., terminal acetylation, amidation,methylation, etc. In some embodiments, proteins can comprise naturalamino acids, non-natural amino acids, synthetic amino acids, andcombinations thereof. The term “peptide” is generally used to refer to apolypeptide having a length of less than about 100 amino acids, lessthan about 50 amino acids, less than 20 amino acids, or less than 10amino acids. In some embodiments, proteins are antibodies, antibodyfragments, biologically active portions thereof, and/or characteristicportions thereof.

Protein: The term “protein” as used herein refers to one or morepolypeptides that function as a discrete unit. If a single polypeptideis the discrete functioning unit and does not require permanent ortemporary physical association with other polypeptides in order to formthe discrete functioning unit, the terms “polypeptide” and “protein” canbe used interchangeably. If the discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” can be used to refer to the multiple polypeptidesthat are physically associated and function together as the discreteunit. In some embodiments, proteins can include moieties other thanamino acids (e.g., glycoproteins, proteoglycans, etc.) and/or can beotherwise processed or modified. Those of ordinary skill in the art willappreciate that in some embodiments the term “protein” can refer to acomplete polypeptide chain as produced by a cell (e.g., with or withouta signal sequence), and/or to a form that is active within a cell (e.g.,a truncated or complexed form). In some embodiments where a protein iscomprised of multiple polypeptide chains, such chains can be covalentlyassociated with one another, for example by one or more disulfide bonds,or can be associated by other means.

Reference: as used herein in a scientific context describes a standardor control relative to which a comparison is performed. For example, insome embodiments, an agent, animal, individual, population, sample,sequence or value of interest is compared with a reference or controlagent, animal, individual, population, sample, sequence or value. Insome embodiments, a reference or control is tested and/or determinedsubstantially simultaneously with the testing or determination ofinterest. In some embodiments, a reference or control is a historicalreference or control, optionally embodied in a tangible medium.Typically, as would be understood by those skilled in the art, areference or control is determined or characterized under comparableconditions or circumstances to those under assessment. Those skilled inthe art will appreciate when sufficient similarities are present tojustify reliance on and/or comparison to a particular possible referenceor control.

Sample: As used herein, the term “sample” typically refers to abiological sample obtained or derived from a source of interest, asdescribed herein. In some embodiments, a source of interest comprises anorganism, such as an animal or human. In some embodiments, a biologicalsample is or comprises biological tissue or fluid. In some embodiments,a biological sample can be or comprise bone marrow; blood; blood cellsof any type (e.g., T cells); ascites; tissue or fine needle biopsysamples; cell-containing body fluids; free floating nucleic acids;sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleuralfluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs;oral swabs; nasal swabs; washings or lavages such as a ductal lavages orbroncheoalveolar lavages; aspirates; scrapings; bone marrow specimens;tissue biopsy specimens; surgical specimens; feces, other body fluids,secretions, and/or excretions; and/or cells therefrom, etc. In someembodiments, a biological sample is or comprises cells obtained from anindividual. In some embodiments, obtained cells are or include cellsfrom an individual from whom the sample is obtained. In someembodiments, a sample is a “primary sample” obtained directly from asource of interest by any appropriate means. For example, in someembodiments, a primary biological sample is obtained by methods selectedfrom the group consisting of biopsy (e.g., fine needle aspiration ortissue biopsy), surgery, collection of body fluid (e.g., blood, lymph,feces etc.), etc. In some embodiments, as will be clear from context,the term “sample” refers to a preparation that is obtained by processing(e.g., by removing one or more components of and/or by adding one ormore agents to) a primary sample. For example, filtering using asemi-permeable membrane. Such a “processed sample” can comprise, forexample nucleic acids or proteins extracted from a sample or obtained bysubjecting a primary sample to techniques such as amplification orreverse transcription of mRNA, isolation and/or purification of certaincomponents, etc.

Specific binding: as used herein, refers to a binding agent's ability todiscriminate between possible partners, in the environment in whichbinding is to occur. A binding agent that interacts with one particulartarget when other potential targets are present is said to “bindspecifically” to the target with which it interacts. In someembodiments, specific binding is assessed by detecting or determiningdegree of association between the binding agent and its partner; in someembodiments, specific binding is assessed by detecting or determiningdegree of dissociation of a binding agent-partner complex; in someembodiments, specific binding is assessed by detecting or determiningability of the binding agent to compete an alternative interactionbetween its partner and another entity. In some embodiments, specificbinding is assessed by performing such detections or determinationsacross a range of concentrations.

Subject: By “subject” is meant a mammal (e.g., a human, in someembodiments including prenatal human forms). In some embodiments, asubject is suffering from a relevant disease, disorder or condition. Insome embodiments, a subject is susceptible to a disease, disorder, orcondition. In some embodiments, a subject displays one or more symptomsor characteristics of a disease, disorder or condition. In someembodiments, a subject does not display any symptom or characteristic ofa disease, disorder, or condition. In some embodiments, a subject issomeone with one or more features characteristic of susceptibility to orrisk of a disease, disorder, or condition. In some embodiments, asubject is a patient. In some embodiments, a subject is an individual towhom diagnosis and/or therapy is, will and/or has been administered.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Variant: As used herein, the term “variant” refers to an entity thatshows significant structural identity with a reference entity butdiffers structurally from the reference entity in the presence or levelof one or more chemical moieties as compared with the reference entity.In many embodiments, a variant also differs functionally from itsreference entity. In general, whether a particular entity is properlyconsidered to be a “variant” of a reference entity is based on itsdegree of structural identity with the reference entity. As will beappreciated by those skilled in the art, any biological or chemicalreference entity has certain characteristic structural elements. Avariant, by definition, is a distinct chemical entity that shares one ormore such characteristic structural elements. To give but a fewexamples, a small molecule can have a characteristic core structuralelement (e.g., a macrocycle core) and/or one or more characteristicpendent moieties so that a variant of the small molecule is one thatshares the core structural element and the characteristic pendentmoieties but differs in other pendent moieties and/or in types of bondspresent (e.g., single vs double, E vs Z, etc.) within the core, apolypeptide can have a characteristic sequence element comprised of aplurality of amino acids having designated positions relative to oneanother in linear or three-dimensional space and/or contributing to aparticular biological function, a nucleic acid can have a characteristicsequence element comprised of a plurality of nucleotide residues havingdesignated positions relative to on another in linear orthree-dimensional space. For example, a variant polypeptide can differfrom a reference polypeptide as a result of one or more differences inamino acid sequence and/or one or more differences in chemical moieties(e.g., carbohydrates, lipids, etc.) covalently attached to thepolypeptide backbone. In some embodiments, a variant polypeptide showsan overall sequence identity with a reference polypeptide that is atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99%. Alternatively, or additionally, in some embodiments, avariant polypeptide does not share at least one characteristic sequenceelement with a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a variant polypeptide shares one or more of the biological activities ofthe reference polypeptide. In some embodiments, a variant polypeptidelacks one or more of the biological activities of the referencepolypeptide. In some embodiments, a variant polypeptide shows a reducedlevel of one or more biological activities as compared with thereference polypeptide. In many embodiments, a polypeptide of interest isconsidered to be a “variant” of a parent or reference polypeptide if thepolypeptide of interest has an amino acid sequence that is identical tothat of the parent but for a small number of sequence alterations atparticular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% of the residues in the variant are substitutedas compared with the parent. In some embodiments, a variant has 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2,or 1) number of substituted functional residues (i.e., residues thatparticipate in a particular biological activity). Furthermore, a varianttypically has not more than 5, 4, 3, 2, or 1 additions or deletions, andoften has no additions or deletions, as compared with the parent.Moreover, any additions or deletions are typically fewer than about 25,about 20, about 19, about 18, about 17, about 16, about 15, about 14,about 13, about 10, about 9, about 8, about 7, about 6, and commonly arefewer than about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent or reference polypeptide is one found in nature.In some embodiments, the variant is engineered. In some embodiments thevariant is naturally occurring. As will be understood by those ofordinary skill in the art, a plurality of variants of a particularpolypeptide of interest can be found in nature.

Vector: as used herein, refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector (e.g., a lentiviral vector or a gammaretroviral vector), wherein additional DNA segments can be ligated intothe viral genome. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) can beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors.”

Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques can beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures can be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

Zinc finger protein: As used herein, the term “zinc finger protein”refers to a polypeptide that contains a “zinc finger domain” throughwhich it is capable of binding to nucleic acids (e.g., DNA). The term“zinc finger domain” refers to an individual “finger”, which comprises aββα-fold stabilized by a zinc ion. Each zinc finger domain typicallyincludes approximately 30 amino acids. Zinc finger domains are largelystructurally independent and can retain their structure and function indifferent environments.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present disclosure areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present disclosure, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the present disclosure will become apparent to those skilled in theart from the detailed description.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure provides, antibodies that bind TALEN proteinsand/or the FokI catalytic DNA cleavage domain and uses thereof

As used herein, antibodies include, but are not limited to antibodybinding regions that are immunologically functional fragments. The term“immunologically functional fragment” (or “fragment”) of an antibody isa species of antibody comprising a portion (regardless of how thatportion is obtained or synthesized) of an antibody that lacks at leastsome of the amino acids present in a full-length chain but which isstill capable of specifically binding to a target antigen (e.g., bindingto FokI and/or TALEN proteins). Such fragments are biologically activein that they bind to the target antigen and can compete with otherantigen binding molecules, including intact antibodies, for binding to agiven epitope. In some embodiments, the fragments are neutralizingfragments. In some embodiments, the fragments can block or reduce theactivity of the target antigen (e.g., blocking effect). In someembodiments, the fragments can antagonize the activity the targetantigen.

In specific embodiments, an anti-TALEN antibody of the instantdisclosure is an antibody identified herein as clones 2299.2G3.C6,2299.5D4.C8, 2299.6B3.B8.B6, 2299.6G5.C7, or 2299.7F11.B4. and eachcomprises the heavy and light chain amino acid, coding, variable, andCDR sequences, as provided and labeled herein. In some embodiments, theclones 2299.2G3.C6, 2299.5D4.C8, 2299.6B3.B8.B6, 2299.6G5.C7, or2299.7F11.B4 specifically bind a molecule comprising a FokI catalyticdomain.

Immunologically functional immunoglobulin fragments include, but are notlimited to, scFv fragments, Fab fragments (Fab′, F(ab′)₂, and the like),one or more complementarity determining regions (“CDRs”), a diabody(heavy chain variable domain on the same polypeptide as a light chainvariable domain, connected via a short peptide linker that is too shortto permit pairing between the two domains on the same chain), domainantibodies, bivalent antigen binding domains (comprises two antigenbinding sites), multispecific antigen binding domains, and single-chainantibodies. These fragments can be derived from any mammalian source,including but not limited to human, mouse, rat, camelid or rabbit. Aswill be appreciated by one of skill in the art, an antigen bindingmolecule can include non-protein components.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by the 3hypervariable regions (CDRs). The CDRs from the two chains of each pairtypically are aligned by the framework regions, which can enable bindingto a specific epitope. From N-terminal to C-terminal, both light andheavy chain variable regions typically comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. By convention, CDR regions in the heavychain are typically referred to as HC CDR1, CDR2, and CDR3. The CDRregions in the light chain are typically referred to as LC CDR1, CDR2,and CDR3.

In some embodiments, anti-TALEN antibodies comprise one or morecomplementarity binding regions (CDRs) present in the full-length lightor heavy chain of an antibody, and in some embodiments comprise a singleheavy chain and/or light chain or portion thereof. These fragments canbe produced by recombinant DNA techniques or can be produced byenzymatic or chemical cleavage of antigen binding domains, includingintact antibodies.

In some embodiments, the antigen binding domain is an antibody offragment thereof, including one or more of the complementaritydetermining regions (CDRs) thereof. In some embodiments, the antigenbinding domain is a single chain variable fragment (scFv), comprisinglight chain CDRs CDR1, CDR2 and CDR3, and heavy chain CDRs CDR1, CDR2and CDR3.

The assignment of amino acids to each of the framework, CDR, andvariable domains is typically in accordance with numbering schemes ofKabat numbering (see, e.g., Kabat et al. in Sequences of Proteins ofImmunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md.1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol196: 901-917; Al-Lazikani et al., (1997) J Mol Biol 273: 927-948;Chothia et al., (1992) J Mol Biol 227: 799-817; Tramontano et al.,(1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No. 7,709,226), contactnumbering, or the AbM scheme (Antibody Modeling program, OxfordMolecular).

Accordingly, in some embodiments, the CDRs of the anti-TALEN antibodiespresented herein are numbered according to the Kabat numbering scheme.In other embodiments, the CDRs of the anti-TALEN antibodies presentedherein are numbered according to the Chothia numbering scheme. In otherembodiments, the CDRs of the anti-TALEN antibodies presented herein arenumbered according to the contact numbering scheme. In otherembodiments, the CDRs of the anti-TALEN antibodies presented herein arenumbered according to the AbM numbering scheme.

Humanized antibodies of the anti-TALEN antibodies described herein canbe prepared by known techniques. In some embodiments, a humanizedmonoclonal antibody comprises the variable domain of an anti-TALENantibody (or all or part of the antigen binding site thereof) and aconstant domain derived from a human antibody. Alternatively, ahumanized antibody fragment can comprise an antigen binding site of amurine or rabbit monoclonal antibody and a variable domain fragment(lacking the antigen binding site) derived from a human antibody.Procedures for the production of engineered monoclonal antibodiesinclude those described in Riechmann et al., (1988) Nature 332:323, Liuet al., (1987) Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al., (1989)Bio/Technology 7:934, and Winter et al., (1993) TIPS 14:139. In someembodiments, the chimeric antibody is a CDR grafted antibody. Techniquesfor humanizing antibodies are discussed in, e.g., U.S. Pat. Nos.5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557; Padlan et al.,(1995) FASEB J. 9:133-39; Tamura et al., (2000) J. Immunol. 164:1432-41;Zhang et al., (2005) Mol. Immunol. 42(12):1445-1451; Hwang et al.,Methods. (2005) 36(1):35-42; Dall'Acqua et al., (2005) Methods36(1):43-60; and Clark, (2000) Immunology Today 21(8):397-402.

Variants of the anti-TALEN antibodies are also within the scope of thedisclosure, e.g., variable light and/or variable heavy chains that eachhave at least 70-80%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99%identity to the amino acid sequences of the antigen binding domainsequences described herein. In some embodiments, the anti-idiotypeantibody is at least about 75%, at least about 85%, at least about 85%,at least about 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or 100% identical toa heavy chain variable region sequence provided in Table 1a and/or alight chain variable sequence provided in Table 1b.

In some instances, such molecules include at least one heavy chain andone light chain, whereas in other instances the variant forms containtwo variable light chains and two variable heavy chains (or subpartsthereof). A skilled artisan will be able to determine suitable variantsof the anti-TALEN antibodies as set forth herein using well-knowntechniques. In certain embodiments, one skilled in the art can identifysuitable areas of the molecule that can be changed without destroyingantigen binding activity by targeting regions not believed to beimportant for activity.

An anti-TALEN antibody of the present disclosure can also be a fullyhuman monoclonal antibody. Fully human monoclonal antibodies can begenerated by any number of techniques with which those having ordinaryskill in the art will be familiar. Such methods include, but are notlimited to, Epstein Barr Virus (EBV) transformation of human peripheralblood cells (e.g., containing B lymphocytes), in vitro immunization ofhuman B-cells, fusion of spleen cells from immunized transgenic micecarrying inserted human immunoglobulin genes, isolation from humanimmunoglobulin V region phage libraries, or other procedures as known inthe art and based on the disclosure herein.

An anti-TALEN antibody that specifically binds to a TALEN protein and/orthe FokI catalytic domain is said to be “selective” when it binds to onetarget more tightly than it binds to a second target. An anti-TALENantibody that specifically binds to a TALEN protein and/or the FokIcatalytic domain is said to “specifically bind” its target antigen(e.g., TALEN protein and/or the FokI catalytic domain) when thedissociation constant (Kd) is in the nanomolar range (e.g., ˜1 nM). Theantigen binding domain specifically binds antigen with “high affinity”when the Kd is 1-5 nM, and with “very high affinity” when the Kd is0.1-0.5 nM. In one embodiment, the antigen binding domain has a Kd of ˜1nM. In one embodiment, the off-rate is <1×10⁻⁵. In other embodiments,the antigen binding domains will bind to TALEN proteins and moleculescomprising FokI catalytic domain with a Kd of between about 1×10⁻⁷ M and1×10⁻¹² M, and in yet another embodiment the antigen binding domainswill bind with a Kd between about 1×10⁻⁵ and 1×10⁻¹².

As provided herein, the anti-TALEN antibodies of the present disclosurespecifically bind TALEN proteins and/or the FokI nuclease domain (e.g.,SEQ ID NO: 1).

In certain embodiments, the anti-TALEN antibodies of the presentdisclosure bind TALEN proteins and/or the FokI catalytic domain with aKD of less than 1×10⁻⁶ M, less than 1×10⁻⁷ M, less than 1×10⁻⁸ M, orless than 1×10⁻⁹ M. In one particular embodiment, the anti-TALENantibodies bind TALEN proteins and/or the FokI catalytic domain with aKD of less than 1×10⁻⁷ M. In another embodiment, the anti-TALENantibodies bind TALEN proteins and/or the FokI catalytic domain with aKD of less than 1×10⁻⁸ M. In some embodiments, the anti-TALEN antibodiesbind TALEN proteins and/or the FokI catalytic domain with a Kd of about1×10⁻⁷ M, about 2×10⁻⁷ M, about 3×10⁻⁷ M, about 4×10⁻⁷ M, about 5×10⁻⁷M, about 6×10⁻⁷ M, about 7×10⁻⁷ M, about 8×10⁻⁷ M, about 9×10⁻⁷ M, about1×10⁻⁸ M, about 2×10⁻⁸ M, about 3×10⁻⁸ M, about 4×10⁻⁸ M, about 5×10⁻⁸M, about 6×10⁻⁸ M, about 7×10⁻⁸ M, about 8×10⁻⁸ M, about 9×10⁻⁸ M, about1×10⁻⁹ M, about 2×10⁻⁹ M, about 3×10⁻⁹ M, about 4×10⁻⁹ M, about 5×10⁻⁹M, about 6×10⁻⁹ M, about 7×10⁻⁹ M, about 8×10⁻⁹ M, about 9×10⁻⁹ M, about1×10⁻¹⁰ M, or about 5×10⁻¹⁰ M. In certain embodiments, the Kd iscalculated as the quotient of K_(off)/K_(on), and the K_(on) and K_(off)are determined using a monovalent antibody, such as a Fab fragment, asmeasured by, e.g., Biacore® surface plasmon resonance technology. Inother embodiments, the Kd is calculated as the quotient ofK_(off)/K_(on), and the K_(on) and K_(off) are determined using abivalent antibody, such as a Fab fragment, as measured by, e.g.,Biacore® surface plasmon resonance technology.

In some embodiments, the anti-TALEN antibodies bind TALEN proteinsand/or the FokI catalytic domain with an association rate (k_(on)) ofless than 1×10⁻⁴ M⁻¹ s⁻¹, less than 2×10 ⁻⁴ M⁻¹ s⁻, less than 3'10⁻⁴ M⁻¹s⁻¹, less than 4×10⁻⁴ M⁻¹ s⁻¹, less than 5×10⁻⁴ M⁻¹ s⁻¹, less than7×10⁻⁴ M⁻¹ s⁻¹, less than 8×10⁻⁴ M⁻¹ s⁻¹, less than 9×10⁻⁴ M⁻¹ s⁻¹, lessthan 1×10⁻⁵ M⁻¹ s⁻¹, less than 2×10⁻⁵ M⁻¹ s⁻¹, less than 3×10⁻⁵ M⁻¹ s⁻¹,less than 4×10⁻⁵ M⁻¹ s⁻¹, less than 5×10⁻⁵ M⁻¹ s⁻¹, less than 6×10⁻⁵ M⁻¹s⁻¹, less than 7×10⁻⁵ M⁻¹ s⁻¹, less than 8×10⁻⁵ M⁻¹ s⁻¹, less than9×10⁻⁵ M⁻¹ s⁻¹, less than 1×10⁻⁶ M⁻¹ s⁻¹, less than 2×10⁻⁶ M⁻¹ s⁻¹, lessthan 3×10⁻⁶ M⁻¹ s⁻¹, less than 4×10⁻⁶ M⁻¹ s⁻¹, less than 5×10⁻⁶ M⁻¹ s⁻¹,less than 6×10⁻⁶ M⁻¹ s⁻¹, less than 7×10⁻⁶ M⁻¹ s⁻¹, less than 8×10⁻⁶ M⁻¹s⁻¹, less than 9×10⁻⁶ M⁻¹ s⁻¹, or less than 1×10⁻⁷ M⁻¹ s⁻¹. In certainembodiments, the k_(on) can be determined using a monovalent antibody,such as a Fab fragment, as measured by, e.g., BIAcore® surface plasmonresonance technology. In other embodiments, the k_(on) is determinedusing a bivalent antibody as measured by, e.g., BIAcore® surface plasmonresonance technology.

In some embodiments, the anti-TALEN antibodies bind TALEN proteinsand/or the FokI catalytic domain with an dissociation rate (k_(off)) ofless than 1×10⁻² s⁻¹, less than 2×10⁻² s⁻¹, less than 3×10⁻² s⁻¹, lessthan 4×10⁻² s⁻¹, less than 5×10⁻² s⁻¹, less than 6×10⁻² s⁻¹, less than7×10⁻² s⁻¹, less than 8×10⁻² s⁻¹, less than 9×10⁻² s⁻¹, less than 1×10⁻³s⁻¹, less than 2×10⁻³ s⁻¹, less than 3×10⁻³ s⁻¹, less than 4×10⁻³ s⁻¹,less than 5×10⁻³ s⁻¹, less than 6×10⁻³ s⁻¹, less than 7×10⁻³ s⁻¹, lessthan 8×10⁻³ s⁻¹, less than 9×10⁻³ s⁻¹, less than 1×10⁻⁴ s⁻¹, less than2×10⁻⁴ s⁻¹, less than 3×10⁻⁴ s⁻¹, less than 4×10⁻⁴ s⁻¹, less than 5×10⁻⁴s⁻¹, less than 6×10⁻⁴ s⁻¹ less than 7×10⁻⁴ s⁻¹, less than 8×10⁻⁴ s⁻¹,less than 9×10⁻⁴ s⁻¹, less than 1×10⁻⁵ s⁻¹, or less than 5×10⁻⁴ s⁻¹. Incertain embodiments, the k_(off) is determined using a monovalentantibody, such as a Fab fragment, as measured by, e.g., BIAcore® surfaceplasmon resonance technology. In other embodiments, the k_(off) isdetermined using a bivalent antibody as measured by, e.g., BIAcore®surface plasmon resonance technology.

Provided herein anti-TALEN antibodies bind TALEN proteins and/or theFokI catalytic domain, comprising a variable heavy chain (VH), whereinthe amino acid sequence or polynucleotide sequence of the VH is selectedfrom the VH sequences presented in Table 1a. The anti-TALEN antibodiesthat specifically bind to the FokI catalytic domain, comprising avariable heavy chain (VH), wherein the amino acid sequence orpolynucleotide sequence of the VH is selected from the VH sequencespresented in Table 1a. In some embodiments, the VH amino acid sequenceis at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or about100% identical to a VH sequence presented in Table la. Kabat CDRdefinitions are shown in bold and Chothia CDR definitions areunderlined.

TABLE 1a Heavy Chain Variable Regions (VH) SEQ ID Clone VH Sequence NO:Amino Acid Sequence 2299.2G3.C6 QIQLVQSGPELKKPGETVKISCKASGYTFTDY SIHWVKQ3 APGEGLKWMAWI NTETGA PTFADDFKGRLALSLETSANT AYLQINNLKHEDTATYFCAKEGGFYFYAMDY WGQGTSVT VSS 2299.5D4.C8 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSIHWVKQ 3 APGEGLKWMAWI NTETGA PTFADDFKGRLALSLETSANT AYLQINNLKHEDTATYFCAKEGGFYFYAMDY WGQGTSVT VSS 2299.6B3.B8.B6 QIQLVQSGPELKKPGETVKISCKASGYAFTDYSIHWMKQ 4 APGEGLQWMGWI NTETAK PAFGDDFKGRFAFSLETSANT AHLHINNLRTEDTATYFCAKEGGFYFYAMDY WGQGTSVT VSS 2299.6G5.C7 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSIHWVKQ 5 APGKGLKWMGWI NTETGA PTFADDFKGRFAFSLETSAST AFLQINNLKNEDTATYFCAKEGGFYFYAMDY WGQGTSLT VSS 2299.7F11.B4 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSIHWVKQ 6 APGKGLKWMAWI NTETGD PTYEDDFKGRFAFSLETSAST AYLQINNLKNEDTATYFCSKEGGFYFYAMDY WGQGTSVT VSS Polynucleotide Sequence 2299.2G3.C6CAGATCCAGTTGGTGCAGTCTGGACCTGAGTTGAAGAAG 29CCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATACACTGGGTGAAGCAGGCTCCAGGAGAGGGTTTAAAGTGGATGGCCTGGATAAACACTGAGACTGGTGCGCCAACATTTGCAGATGACTTCAAGGGACGGTTAGCCCTCTCTTTGGAGACTTCTGCCAACACTGCCTATTTGCAGATCAACAACCTCAAACATGAAGACACGGCTACATATTTCTGTGCTAAAGAAGGTGGTTTCTACTTTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACC GTCTCCTCA 2299.5D4.C8CAGATCCAGTTGGTGCAGTCTGGACCTGAGTTGAAGAAG 31CCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATACACTGGGTGAAGCAGGCTCCAGGAGAGGGTTTAAAGTGGATGGCCTGGATAAACACTGAGACTGGTGCGCCAACATTTGCAGATGACTTCAAGGGACGGTTAGCCCTCTCTTTGGAGACCTCTGCCAACACTGCCTATTTGCAGATCAACAACCTCAAACATGAAGACACGGCAACATATTTCTGTGCTAAAGAAGGTGGTTTCTACTTTTATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACC GTCTCTTCA 2299.6B3.B8.B6CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAG 32CCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATGCCTTCACAGACTATTCAATACACTGGATGAAGCAGGCTCCAGGAGAGGGTCTACAGTGGATGGGCTGGATAAACACTGAGACTGCTAAGCCAGCATTTGGAGATGACTTCAAGGGACGGTTTGCCTTTTCTTTGGAAACCTCTGCCAACACTGCCCATTTGCACATCAACAACCTCAGAACTGAAGACACGGCTACATATTTCTGTGCTAAAGAAGGTGGTTTCTACTTTTATGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACC GTCTCCTCA 2299.6G5.C7CAGATCCAGTTGGTGCAGTCTGGACCTGAGTTGAAGAAG 33CCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATACACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGCGCCAACATTTGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTTTTTGCAGATCAACAACCTCAAAAATGAAGACACGGCTACATATTTCTGTGCTAAGGAAGGTGGTTTCTACTTTTACGCTATGGACTACTGGGGTCAAGGAACCTCGCTCACC GTCTCCTCA 2299.7F11.B4CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAG 34CCTGGAGAGACAGTCAAGATCTCCTGCAAGGCCTCTGGTTATACCTTCACAGACTACTCAATACACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGCCTGGATAAACACTGAGACTGGTGACCCAACATATGAAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTTCTAAAGAAGGTGGTTTCTACTTTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACC GTCTCCTCA

Provided herein are anti-TALEN antibodies that specifically bind to theFok1 catalytic domain, comprising a variable light chain (VL), whereinthe amino acid sequence or polynucleotide sequence of the VL is selectedfrom the VL sequences presented in Table 1b. In some embodiments, the VLamino acid sequence is at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% identical to a VL sequence presented in Tablelb. Kabat CDR definitions are shown in bold and Chothia CDR definitionsare underlined.

TABLE 1b Light Chain Variable Regions (VL) SEQ ID Clone VL Sequence NO:Amino Acid Sequence 2299.2G3.C6 DIVMSQSPSSRAVSAGEKVTMSCKSSQSLLSSRTRKNYLA WYQ 7 QKPGQAPKMLIS WASTREF GVPDRFTGSGFGTDFTLTISSVQGEDLAVYYC KQSYNFRT FGGGTKLEIK 2299.5D4.C8 DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLSSRTRKNYLA WYQ 8 QKPGQAPKLLIS WASTRES GVPDRFTGSGSGTEFTLTISSVQAEDLAVYYC KQSYNLRT FGGGTKLEIK 2299.6B3.B8.B6 DIVMSQSPSSLAVSAGEKVTMNCKSSQSLLSSRTRKNYLA WYQ 9 QKPGQSPKLLIY WASTRDS GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSYNFRT FGGGTKLEIK 2299.6G5.C7 DIVMSQSPSSLAVSTGEKVTMSCKSSQSLLSSRTRKNYLA WYQ 10 QKPGQAPKLLIS WASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSFNLRT FGGGTKLEIK 2299.7F11.B4 DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLSSRTRKNYLA WYQ 11 QKPGQSPKLLIK WASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSYNLRT FGGGTKLEIK Polynucleotide Sequence 2299.2G3.C6GACATTGTGATGTCACAGTCTCCATCCTCCCGGGCTGTGTCAG 30CAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAGCAGTCGAACCCGAAAGAACTACTTGGCTTGGTACCAACAGAAACCAGGGCAGGCTCCTAAAATGCTGATTTCCTGGGCATCCACTAGGGAATTTGGGGTCCCTGATCGCTTCACAGGCAGTGGATTTGGGACAGATTTCACTCTCACCATTAGCAGTGTGCAGGGTGAGGACCTGGCAGTTTATTACTGCAAACAATCTTATAATTTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 2299.5D4.C8GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAG 35CAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGCTCAGCAGTCGAACCCGAAAGAACTACTTGGCTTGGTACCAACAGAAACCAGGGCAGGCTCCTAAACTGCTGATCTCCTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGTGTGCAGGCTGAGGACCTGGCAGTTTATTACTGCAAACAATCTTATAATCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 2299.6B3.B8.B6GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAG 36CAGGAGAGAAGGTCACTATGAACTGCAAATCCAGTCAGAGTCTGCTCAGCAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCTTCCACTAGGGACTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCAGAGGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATTTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 2299.6G5.C7GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTATCAA 37CAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAGCAGTCGAACCCGAAAGAACTACTTGGCTTGGTACCAACAGAAACCAGGGCAGGCTCCTAAACTGCTGATCTCCTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAACAATCTTTTAATCTTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAA 2299.7F11.B4GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAG 38CAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAGCAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTAAATGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

Provided herein are anti-TALEN antibodies that specifically bind to theFok1 catalytic domain, wherein anti-TALEN antibodies comprise a variableheavy chain (VH) and a variable light chain (VL), wherein the amino acidsequence or polynucleotide sequence of the VH is selected from the VHsequences presented in Table 1a; and wherein the amino acid sequence orpolynucleotide sequence of the VL is selected from the VL sequencespresented in Table 1b.

In some embodiments, the anti-TALEN antibodies that specifically bind tothe Fok1 catalytic domain, comprise a VH CDR 1, CDR2, and CDR3 of a VHsequence presented in Table 1a. In some embodiments, the VH CDR 1, CDR2,and CDR3 are selected from a CDR sequence presented in Table 1c.

-   -   (ii)

TABLE 1c Heavy Chain CDRs CDR1 VH SEQ CDR1 VH SEQ Sequence ID SequenceID Clone (Chothia) NO: (Kabat) NO: 2299.2G3.C6 GYTFTDY 12 DYSIH 142299.5D4.C8 GYTFTDY 12 DYSIH 14 2299.6B3.B8.B6 GYAFTDY 13 DYSIH 142299.6G5.C7 GYTFTDY 12 DYSIH 14 2299.7F11.B4 GYTFTDY 12 DYSIH 14 CDR2 VHSEQ CDR2 VH SEQ Sequence ID Sequence ID Clone (Chothia) NO: (Kabat) NO:2299.2G3.C6 NTETGA 15 WINTETGAPTF 18 ADDFKG 2299.5D4.C8 NTETGA 15WINTETGAPTF 18 ADDFKG 2299.6B3.B8.B6 NTETAK 16 WINTETAKPAF 19 GDDFKG2299.6G5.C7 NTETGA 15 WINTETGAPTF 18 ADDFKG 2299.7F11.B4 NTETGD 17WINTETGDPTY 20 EDDFKG CDR3 VH SEQ CDR3 VH SEQ Sequence ID Sequence IDClone (Chothia) NO: (Kabat) NO: 2299.2G3.C6 EGGFYFYAMDY 21 EGGFYFYAMDY21 2299.5D4.C8 EGGFYFYAMDY 21 EGGFYFYAMDY 21 2299.6B3.B8.B6 EGGFYFYAMDY21 EGGFYFYAMDY 21 2299.6G5.C7 EGGFYFYAMDY 21 EGGFYFYAMDY 21 2299.7F11.B4EGGFYFYAMDY 21 EGGFYFYAMDY 21

In some embodiments, anti-TALEN antibodies that specifically bind to theFok1 catalytic domain, comprise a VL CDR 1, CDR2, and CDR3 of a VLsequence presented in Table 1b. In some embodiments, the VH CDR 1, CDR2,and CDR3 are selected from a CDR sequence presented in Table 1d.

TABLE 1d Light Chain CRs SEQ ID Clone NO: CDR1 VL Sequence(Kabat and Chothia) 2299.2G3.C6 KSSQSLLSSRTRKNYLA 22 2299.5D4.C8KSSQSLLSSRTRKNYLA 22 2299.6B3.B8.B6 KSSQSLLSSRTRKNYLA 22 2299.6G5.C7KSSQSLLSSRTRKNYLA 22 2299.7F11.B4 KSSQSLLSSRTRKNYLA 22 CDR2 VL Sequence(Kabat and Chothia) 2299.2G3.C6 WASTREF 23 2299.5D4.C8 WASTRES 242299.6B3.B8.B6 WASTRDS 25 2299.6G5.C7 WASTRES 24 2299.7F11.B4 WASTRES 24CDR3 VL Sequence (Kabat and Chothia) 2299.2G3.C6 KQSYNFRT 26 2299.5D4.C8KQSYNLRT 27 2299.6B3.B8.B6 KQSYNFRT 26 2299.6G5.C7 KQSFNLRT 282299.7F11.B4 KQSYNLRT 27

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry,computational chemistry, protein engineering, antibody structure andfunction, cell culture, recombinant DNA and related fields as are withinthe skill of the art. These techniques are fully explained in theliterature. See, for example, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press,1989 and Third edition, 2001 and subsequent editions; Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, AcademicPress, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Thirdedition, Academic Press, San Diego, 1998 and subsequent editions.

Genome Editing Technologies Transcription Activator-Like EffectorNucleases

Transcription activator-like effector nucleases (TALEN) typically referto restriction enzymes that can be engineered to cut specific sequencesof DNA. They generally include a TAL effector DNA-binding domain and aDNA cleavage domain, i.e., a nuclease which cuts DNA strands.Transcription activator-like effectors (TALEs) can be engineered to bindpractically any desired DNA sequence. Thus, TALENs can be introducedinto cells, for use in gene editing or for genome editing in situ.

Various non-specific DNA cleavage domain can be used in TALEN constructsincluding, but not limited to, wild-type FokI cleavage domain, portionsof the FokI endonuclease, FokI cleavage domain variants such as thosewith mutations designed to improve cleavage specificity and cleavageactivity. In some embodiments, a FokI domain functions as a dimer,involving two constructs with DNA binding domains for sites in a targetgenome with proper orientation and spacing. In some embodiments, theTALEN comprises an endonuclease (e.g., FokI) cleavage domain or cleavagehalf-domain. The disclosed antibodies can be used for any TALEN-mediatedgene editing, including targeted integration; methods and compositionsfor nuclease-mediated targeted integration are described inWO2015127439A1, which is incorporated by reference in its entirety.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are protein chimera comprised of a zincfinger-based DNA-binding domain and a DNA-cleavage (i.e. nuclease)domain (e.g., FokI). ZFNs are able to introduce double-strand breaks(DSB; breaks at the same or very close points in both strands of adouble-stranded DNA molecule) at specific locations within a DNAmolecule which can subsequently be used to disable, replace, insert oredit a targeted genomic region.

Zinc fingers are among the most common DNA binding motifs found ineukaryotes. These proteins are classified according to the number andposition of the cysteine and histidine residues available for zinccoordination. A zinc finger consists of two antiparallel β strands, andan a helix. The zinc ion is crucial for the stability of this domaintype—in the absence of the metal ion the domain unfolds as it is toosmall to have a hydrophobic core. One very well-explored subset ofzinc-fingers (the C₂H₂ class) comprises a pair of cysteine residues inthe beta strands and two histidine residues in the alpha helix which areresponsible for binding a zinc ion. The two other classes of zinc fingerproteins are the C₄ and C₆ classes. Zinc fingers are important inregulation because when interacted with DNA and zinc ion, they provide aunique structural motif for DNA-binding proteins. The structure of eachindividual finger is highly conserved and consists of about 30 aminoacid residues, constructed as a ββα fold and held together by the zincion. The α-helix occurs at the C-terminal part of the finger, while theβ-sheet occurs at the N-terminal part.

The DNA-binding domain of a ZFN can be composed of two to eight zincfinger motifs due to their supposed modularity. In some embodiments, aZFN comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs. Each zincfinger motif is typically considered to recognize and bind to athree-base pair sequence and as such, a protein including more zincfingers targets a longer sequence and therefore has a greaterspecificity and affinity to the target site. Methods to engineer zincfinger binding domains to bind to a sequence of choice are well known inthe art. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. Zinc finger engineering methods known in the artinclude, but are not limited to, rational design and various types ofselection. Rational design includes, for example, using databasescomprising triplet (or quadruplet) nucleotide sequences and individualzinc finger amino acid sequences, in which each triplet or quadrupletnucleotide sequence is associated with one or more amino acid sequencesof zinc fingers which bind the particular triplet or quadrupletsequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261.Depending upon the required specifications of the end-product, theincluded zinc fingers can be selected via a parallel, sequential orbipartite technique or through an in vitro cell-based technique.Exemplary selection methods are also known in the art, including phagedisplay and two-hybrid systems, see, U.S. Pat. Nos. 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568;and international patent publications WO 98/37186; WO 98/53057; WO00/27878; and WO 01/88197. Enhancements of binding specificity for zincfinger binding domains have been previously described, for example, inWO 02/077227.

The non-specific nuclease domain of FokI is functionally independent ofits natural DNA-binding domain and therefore can be employed in theconstruction of ZFNs. Since the domain must dimerize to accomplish adouble-strand break it is necessary that a nuclease is also bound to theopposite strand by virtue of another ZFN molecule bound to its targetsequence as shown in the diagram. The two target sites need not be thesame, so long as ZFNs targeting both sites are present. In order to forma dimer, two ZFN molecules must meet with their respective recognitionsites not less than 4-6 base pairs apart but also not so far apart thatthey may not dimerize. While one ZFN molecule binds its target sequenceon one strand, another ZFN molecule binds its target sequence on theopposite strand, as shown in the diagram. The nuclease domains dimerizeand each cleaves its own strand, producing a DSB. FokI can be employedas a homo- or a heterodimer. In some embodiments, FokI is a homodimer.In some embodiments, FokI is a heterodimer.

RNA-Guided FokI Nuclease

RNA-guided FokI nucleases (RFNs) are protein chimera comprised of a Cas9nuclease (Cas9) DNA-binding domain and a DNA-cleavage domain (e.g.,FokI-cleavage domain). RFNs specifically target particular DNA sequencesthrough the use of a guide RNA (gRNA) bearing 17-20 nucleotides oftarget site complementarity at the 5′ end. RFNs require dimerization ofthe FokI DNA cleavage domain to introduce double-strand breaks (DSB;breaks at the same or very close points in both strands of adouble-stranded DNA molecule) at specific locations within a DNAmolecule which can subsequently be used to disable, replace, insert oredit a targeted genomic region.

CRISPR

CRISPR-associated Cas systems have evolved in bacteria and archaea touse short RNAs to guide DNA cleavage complexes to specific nucleotidesequences. The CRISPR/Cas system has been adapted for genome editing ineukaryotic cells. Cas9 unwinds DNA and checks for complementarity to the20 base pair spacer region of the gRNA thus specifically targeting DNAsequences preceding protospacer adjacent motifs (PAM) sequences inmammalian cell genomes. The introduction of DSBs allows for targetsequence alteration through one of two endogenous DNA repairmechanisms—either non-homologous end-joining (NHEJ) or homology-directedrepair (HDR). The CRISPR/Cas system has also been used for generegulation including transcription repression and activation withoutaltering the target sequence. Targeted gene regulation based on theCRISPR/Cas system uses an enzymatically inactive Cas9 (dCas9).Additional Cas9 descriptions and gRNA requirements for programmabledCas9 are incorporated in but not limited to U.S. patent Ser. No.14/536319, Ser. No. 14/320498, and Ser. No. 14/320467

Genome Editing Systems

As described above, TALENs, ZFNs and RFNs can bind to and cleave attargeted sites in a genome, permitting the insertion or deletion ofspecific DNA sequences, notably when a repair template is provided.Selective cutting is achieved through sequence specific targeting by theDNA binding domain (e.g., TAL effector DNA-binding domain) and DNAcleavage by FokI nucleases. After the TALEN construct creates a doublestranded break at the target locus, the homologous recombinationmachinery searches for homology between the damaged chromosome and theextra-chromosomal fragment and copies the sequence of the fragmentbetween the two broken ends of the chromosome, regardless of whether thefragment contains the original sequence, resulting in insertion of thegene of interest into the genome.

In addition to homology-mediated gene editing the TALEN approach canalso be applied to gene editing (knock outs and insertions) mediated bynon-homologous end joining.

In some embodiments, the genome editing system includes 1, 2, 3, 4, 5,6, 7, 8 or more nucleic acid construct(s) encoding a “half-TALEN” whichassemble to form a complete TALEN with targeted FokI-mediated nucleaseactivity. In some embodiments, the genome editing system includes two orfour nucleic acid constructs encoding “half-TALENs” with targetedFokI-mediated nuclease activity. In some embodiments, the nucleic acidsencode dimeric TALENs, or a TALEN comprising two “half-TALENs”.

In some embodiments, a genome editing system includes a polynucleotideconstruct bearing a donor sequence which undergoes genomic insertionafter nuclease cleavage. In some embodiments, a donor sequence will haveone or more homologous region(s) that share identity to a genomicsequence with which recombination is desired. In some embodiments, adonor sequence will have one or more homologous region(s) that have atleast 50% sequence identity to a genomic sequence with whichrecombination is desired. In some embodiments, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 99.9% sequence identity is present over a homologousregion. Any value between 1% and 100% sequence identity can be present,depending upon the length of the donor polynucleotide.

In some embodiments, as part of gene therapy, DNA can be introduced intoselected target cells in a manner such that the donor DNA is expressed,and a therapeutic product encoded thereby is produced. Additionally, oralternatively, donor DNA can in some manner mediate expression of DNAthat encodes the therapeutic product, or it can encode a product, suchas a peptide or RNA that in some manner mediates, directly orindirectly, expression of a therapeutic product. In some embodiments thedonor DNA encodes a therapeutic product for the treatment or preventionof disease or disorder that results from a genetically defect ordeficiency. In some embodiments, the donor DNA encodes a therapeuticproduct for the treatment or prevention of cancer or any other diseaseor disorder that results from a genetic defect or deficiency. In someembodiments, the donor DNA encodes a chimeric antigen receptor (CAR).

Techniques and protocols for genome editing are described in the art.See, for example, US 2005/0215502; US 2007/0134796; WO 2005/084190;WO2014204578, Urnov et al., Nature Reviews Genetics 11, 636-646 (2010);Rahman et al., Human Gene Therapy 22(8): 925-933 (2011); Tsai et al.,Nature Biotechnology (2014) 32: 569-576; and Collins et al., ProceedingsBiological Sciences/The Royal Society, 282(1821):pii 20143003 (2015).For example, genome editing can be performed in vivo or ex vivo (e.g. invitro). In some embodiments, viral and non-viral based gene transfermethods can be used to introduce nucleic acids encoding TALENs, ZFNs orRFNs into mammalian cells or target tissues. Such methods can be used toadminister nucleic acids encoding TALENs, ZFNs or RFNs to cells invitro. In some embodiments, non-viral vector delivery systems includeDNA plasmids, naked nucleic acid, and nucleic acid complexed with adelivery vehicle such as poloxamers or liposomes. In some embodiments,the nucleic acids encoding TALENs, ZFNs or RFNs are transfected viaelectroporation. In some embodiments, the non-viral delivery systemincludes direct transfer of TALEN-, ZFN- or RFN-encoding mRNA, such asby microinjection. Viral vector delivery systems include DNA and RNAviruses, which have either episomal or integrated genomes after deliveryto the cell. Examples of viral vector delivery systems include, but arenot limited to, integrase-deficient lentiviral vectors, adenoviralvectors, and vectors based on adeno-associated virus. In someembodiments, components of the genome editing system are mixed prior toadministering to a subject. In some embodiments, components of thegenome editing system are administered separately. Separateadministration of the genome editing components can be essentiallysimultaneous, sequential or separated by a determined period of time.

Described herein, are highly sensitive and reliable methods fordetecting and/or measuring FokI in a sample. As shown herein, thesemethods can detect FokI at physiological levels.

Detection Moieties

Detection moieties as used herein include moieties of any structurecapable of being detected by any methods known in the art. A detectionmoiety refers to any element, molecule, functional group, compound,fragment or moiety that is detectable.

Detection moieties include, but are not limited to, radionuclides (e.g.,³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y,^(99m)Tc, ¹⁷⁷Lu, ⁸⁹Zr etc.), fluorescent dyes (such as, for examplefluorescein dyes, acridine dyes, SYBR dyes, rhodamine dyes, oxazinedyes, etc.), chemiluminescent agents (such as, for example, acridinumesters, stabilized dioxetanes, and the like), electrochemiluminescentagents (such as, for example, Sulfo Tags), bioluminescent agents (suchas, for example, luciferin), spectrally resolvable inorganic fluorescentsemiconductors nanocrystals (i.e., quantum dots), metal nanoparticles(e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagneticmetal ions, enzymes (such as, for example, horseradish peroxidase,alkaline phosphatase, etc.), colorimetric labels (such as, for example,dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, andproteins for which antisera or monoclonal antibodies are available.

In some embodiments, the anti-FokI antibody is associated with adetection moiety. The anti-FokI antibody can be covalently ornon-covalently associated with the detection moiety. In some embodimentsthe detection moiety is selected from sulfo-tag, horseradish peroxidase,alkaline phosphatase, FITC, digoxigenin, acridan, RPE, luciferin,fluorophores, chromophores, radioisotopes, or biotin.

In some embodiments, the detection moiety is an electrochemiluminescent(ECL) label. In general, ECL-based assays involve the use ofelectrochemiluminescent compounds as labels (referred to herein as ECLlabels). Any compounds capable of electrochemiluminescence can be usedas an ECL labels. Examples of ECL labels include, but are not limitedto, organometallic compounds such as the tris-bipyridyl-ruthenium(RuBpy) moiety where the metal is from, for example, the metals of groupVII and VIII, including Re, Ru, Ir and Os. In some embodiments, the ECLlabel is a Sulfo-Tag label. Species that react with the ECL label in theECL process are referred to herein as ECL coreactants. Commonly usedcoreactants for ECL include tertiary amines (e.g. tripropylamine (TPA)),oxalate, and persulfate.

In some embodiments, the anti-TALEN antibody is associated with adetection moiety. The detection moiety can be associated with theanti-TALEN antibody through covalent conjugation or through non-covalentassociation. Covalent conjugation of the detection moiety to theanti-TALEN antibody can be direct or can be through a linker. In someembodiments the detection moiety is selected from an ECL tag (e.g.,Sulfo-Tag), horseradish peroxidase, alkaline phosphatase, FITC,digoxigenin, acridan, RPE, luciferin, fluorophores, chromophores,radioisotopes, or biotin.

The anti-TALEN antibodies described herein can be used in any assayknown in the art to measure presence or intensity of a detection moietyin immunoassays. In some embodiments, detecting the presence or absenceof a FokI comprising molecule (e.g., a TALEN protein) comprisesmeasurement of electrochemiluminescence, luminescence, fluorescence, orabsorbance by any means known in the art. The detecting step can involvea quantitative measurement, which can be used to determine the amount ofFokI polypeptide in the sample. Measurement(s) to determine the presenceor intensity of FokI can be performed by a hand method or can be carriedout using an apparatus such as an analyzer. For example, in someembodiments, the detection moiety can produce a color output. Methodscan include visual appearance of a colored product and/or an instrumentsuch as an analyzer can be used to detect or quantify the color product.In some embodiments, the detecting step comprises a measurement ofelectrochemiluminescence, which can be performed using a commerciallyavailable instrument.

In some embodiments, the detection moiety is a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, or a hapten. In some embodiments, the detectablelabel is a fluorescent label is selected from the group consisting of anAtto dye, an Alexa Fluor dye, quantum dots, Hydroxycoumarin,Aminocouramin, Methoxycourmarin, Cascade Blue, Pacific Blue, PacificOrange, Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates,PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein,BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine,Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC), APC-Cy7conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP,Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, KusabiraOrange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP,DsRed monomer, DsRed2, mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red,R-phycoerythrin (RPE), B-phycoeryhring (BPE), mCherry, HcRed1, Katusha,P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum,and mRaspberry.

Type-2 Restriction Enzyme FokI

FokI is a bacterial type IIS restriction endonuclease that is naturallyfound in Flavobacterium okeanokoites. Natural FokI consists of anN-terminal DNA-binding domain and a non-specific DNA cleavage domain atthe C-terminus. (UniProt Accession Number P14870) The full length wildtype FokI amino acid sequence is:

(SEQ ID NO: 2) MFLSMVSKIRTFGWVQNPGKFENLKRVVQVFDRNSKVHNEVKNIKIPTLVKESKIQKELVAIMNQHDLIYTYKELVGTGTSIRSEAPCDAIIQATIADQGNKKGYIDNWSSDGFLRWAHALGFIEYINKSDSFVITDVGLAYSKSADGSAIEKEILIEAISSYPPAIRILTLLEDGQHLTKFDLGKNLGFSGESGFTSLPEGILLDTLANAMPKDKGEIRNNWEGSSDKYARMIGGWLDKLGLVKQGKKEFIIPTLGKPDNKEFISHAFKITGEGLKVLRRAKGSTKFTRVPKRVYWEMLATNLTDKEYVRTRRALILEILIKAGSLKIEQIQDNLKKLGFDEVIETIENDIKGLINTGIFIEIKGRFYQLKDHILQFVIPNRGVTKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKENNGEINF

The FokI DNA cleavage domain can function independently of the DNAbinding domain. In some embodiments, a FokI polypeptide consists,consists essentially of or comprises the FokI nuclease domain. In someembodiments, the FokI catalytic domain polypeptide is wild-type aminoacids 439-583 of SEQ ID NO: 2. In some embodiments, the anti-TALENantibodies described herein bind to a molecule comprising SEQ ID NO:1.

(SEQ ID NO: 1) MKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKENNG EINF

In some embodiments, the FokI protein is modified. In some embodiments,the modified FokI protein includes an amino acid sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to wild-type FokIcatalytic domain (SEQ ID NO:1). In some embodiments, the FokI proteincomprises biologically active fragments of FokI. In some embodiments thebiologically active fragment includes the catalytic domain of FokI. Insome embodiments, FokI protein includes the DNA-cleavage domain of FokI.In some embodiments, the FokI protein comprises an amino acid sequencesthat differs from SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, or more amino acids.

Sample Types

Sample types of the present disclosure include any materials that have,are suspected to have, are likely to have, or are confirmed to haveTALEN proteins or molecules comprising a FokI catalytic domain. In someembodiments, the sample is an in vitro sample. In some embodiments, thesample is an in vivo sample. In some embodiments, the sample is ofbiological origin (e.g., a biological sample). Biological samplesinclude all materials that at one time were part of a living organism.In some embodiments, the biological sample is from a mammal. Mammalsinclude, but are not limited to, humans, dogs, cats, cows, pigs, sheep,non-human primates and llamas. Humans can be of any age. The biologicalsample can take any form and include, but are not limited to, cells,tissue, whole blood, plasma, serum, urine, stool, saliva, cord blood,sperm, aqueous or vitreous humor, chorionic villus sample, andcombination thereof. In some embodiments, the sample is a tissue sample,a blood sample, a formalin-fixed sample, a tissue grown ex vivo or cellculture media. In some embodiments, a sample e.g., a serum sample is tobe analyzed for the presence (or absence) or level of TALEN or FokIprotein.

Kits

Also provided herein are kits for use in detecting and/or measuring FokIin a sample (e.g., a biological sample), as described herein. Such kitstypically comprise one or more substances, reagents and/or materials foruse in the methods provided herein. The kit can include instructions forperforming such methods. Kits for detecting and/or measuring FokI caninclude various reagents necessary for the analyzing 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 400, 500, 600, 700, 800, 900, 1000 or more samples. In someembodiments, the kit can allow for the simultaneous or near simultaneousanalysis of a plurality of samples, such as a microplate-type assay kit.In some embodiments, the kit can be disposable, such that reagents andmaterials are intended for a single use and/or to be disposed of afterperformance of the assay.

In some embodiments, a kit for use in detecting and/or measuring FokI ina sample comprises an anti-TALEN antibody described herein andoptionally conjugated to a detection moiety, or can comprise materialsallowing a used of the kit to perform such a conjugation. Substrates ofthe FokI detection kits described herein can take any suitable form thatpermits the association of anti-FokI antibodies described herein andFokI containing complexes. In some embodiments, the substrate iscontained in or part of a microplate. Any suitable detection moietyknown in the art can be used. For example, detection moieties include,but are not limited to, an ECL tag (e.g., Sulfo-Tag), horseradishperoxidase, alkaline phosphatase, FITC, digoxigenin, acridan, RPE,luciferin, fluorophores, chromophores, radioisotopes, or biotin.Further, any suitable methods of detection can be used in conjunctionwith FokI detection kits described herein. The kit may or may notinclude reagents, materials and or instruments for performing thedetection or measurement of signal. In some embodiments, the use of akit can include the detection of signal, which comprises measurement ofelectrochemiluminescence, luminescence, fluorescence, or absorbance.Detection of signal associated with an anti-FokI detection assay can beby any means known in the art. Associated kits may or may not includeinstructions for a specific detection method. In some embodiments, a kitcan refer a user to standard methods of detection. In some embodiments,the kit can refer a user to follow instructions associated with otherinstrumentation for detection.

Representative Uses

In some embodiments, methods and kits of the present disclosure can beused for the evaluation and/or monitoring of cellular or gene therapy,such as CAR T therapy. In some embodiments the cellular or gene therapyis a ZFN-based gene therapy. In some embodiments, the cellular or genetherapy is a RFN-based gene therapy. In some embodiments, the cellulartherapy is a TALEN-based gene therapy. In some embodiments, the cellularor gene therapy comprises the administration of a DNA construct. In someembodiments, the cellular or gene therapy comprises mRNA replacementtherapy. In some embodiments, the cellular or gene therapy comprisescellular replacement therapy, such as Chimeric Antigen Receptor(CAR)-based therapy, which can comprise allogeneic or autologouscellular replacement. Cellular replacement therapy can compriseadministering cells that have been transduced or transfected with anucleic acid construct encoding a FokI protein or a biologically activefragment thereof.

In some embodiments, samples for evaluating and/or monitoring cellularor gene therapy can be obtained prior to the initiation of therapy. Insome embodiments, samples are obtained after a first cellular or genetherapy treatment or dose. In some embodiments, samples are obtainedafter the conclusion of gene therapy. In some embodiments, samples areobtained at specific time points, intervals, or any other metric of timebefore, during or after cellular or gene therapy is performed. In someembodiments, FokI detection is used for assessing potential for cellularor gene therapy.

Methods of Treatment

In some embodiments, the anti-TALEN antibodies of the present disclosurecan be used for the evaluation and/or monitoring of gene therapy orcellular replacement therapy, such as CAR therapy. In some embodiments,the gene therapy comprises administering to a patient a population ofcells engineered with a TALEN protein to express a chimeric antigenreceptor (CAR). The CAR containing immune cells can be used to treatmalignancies involving aberrant expression of biomarkers, includingcancer. In some embodiments, CAR containing immune cells of thedisclosure can be used to treat non-Hodgkins lymphoma such as diffuselarge B-cell lymphoma (DLBCL), leukemia (e.g., CLL or ALL), multiplemyeloma (MM), small cell lung cancer, melanoma, low grade gliomas,glioblastoma, medullary thyroid cancer, carcinoids, dispersedneuroendocrine tumors in the pancreas, bladder and prostate, testicularcancer, and lung adenocarcinomas with neuroendocrine features.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood-born tumors. The term “cancer” refers to disease ofskin tissues, organs, blood, and vessels, including, but not limited to,cancers of the bladder, bone or blood, brain, breast, cervix, chest,colon, endometrium, esophagus, eye, head, kidney, liver, lymph nodes,lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,advanced malignancy, amyloidosis, neuroblastoma, meningioma,hemangiopericytoma, multiple brain metastases, glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, recurrent malignant giolma, anaplastic astrocytoma,anaplastic oligodendroglioma, neuroendocrine tumor, rectaladenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectalcarcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma,karotype acute myeloblastic leukemia, Hodgkin lymphoma, non-Hodgkinlymphoma (NHL), cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma,diffuse large B-Cell lymphoma, low grade follicular lymphoma, malignantmelanoma, malignant mesothelioma, malignant pleural effusionmesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma,gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneousvasculitis, Langerhans cell histiocytosis, leiomyosarcoma,fibrodysplasia ossificans progressive, hormone refractory prostatecancer, resected high-risk soft tissue sarcoma, unrescectablehepatocellular carcinoma, Waldenstrom's macroglobulinemia, smolderingmyeloma, indolent myeloma, fallopian tube cancer, androgen independentprostate cancer, androgen dependent stage IV non-metastatic prostatecancer, hormone-insensitive prostate cancer, chemotherapy-insensitiveprostate cancer, papillary thyroid carcinoma, follicular thyroidcarcinoma, medullary thyroid carcinoma, and leiomyoma. In a specificembodiment, the cancer is metastatic. In another embodiment, the canceris refractory or resistant to chemotherapy or radiation. In a furtherembodiment a cancer has been reduced by a degree determined to be lessthan desire and a re-treatment is determined to be appropriate.

EXAMPLES Example 1: Generation of Anti-TALEN Antibodies

To generate hybridoma cells, wildtype Balb/c mice were immunized withFokI catalytic domain protein including a C-terminal 6xHis tag and GSGSlinker. Using repetitive immunizations multiple sites (REVIMS), cellsfrom immunization day 12 Balb/c mice were fused to obtain hybridomafusion #2296. The resulting antibodies were screened on FokI catalyticdomain for ELISA positives and counter screened using the His-tagantigen. Samples were also screened on full length TALEN proteinincluding a C-terminal 6xHis tag and GSGS linker. Antibodies wereanalyzed by Western Blot and assessed using Biacore® for bindingconfirmation and kinetics.

Antibodies obtained from fusion #2296 resulted in over 30 ELISApositives screened on FokI and full-length TALEN protein. Theseantibodies were negative on His-tag antigen. Biacore® analysis of fusion#2296 antibodies resulted in kD values ranging from 5 nM to 100 nM. TheBiacore® kinetics of fusion #2296 REVIMS Day 12 are shown in Table 2below.

TABLE 2 Biacore ® kinetics of Fusion 2296 Binds FokI Ligand Analytek_(a) (1/Ms) k_(d) (1/s) t_(1/2) (min) K_(D) (nM) or TALEN (Y/N/A)A01_1A8 FokI 5.51E+03 3.51E−04 32.9 63.7 Y A01_1A8 TALEN Y A02_7E12 FokI2.72E+03 4.72E−04 24.5 174 Y A02_7E12 TALEN Y A03_14H5 FokI 5.47E+047.09E−04 16.3 13.0 Y A03_14H5 TALEN Y A05_1G2 FokI 7.87E+05 3.16E−02 0.440.2 Y A05_1G2 TALEN Y B04_22A5 FokI 1.01E+06 4.62E−02 0.3 45.7 YB04_22A5 TALEN Y B05_2G3 FokI 1.18E+04 6.15E−04 18.8 52.1 Y B05_2G3TALEN Y C03_17B5 FokI 2.02E+05 5.01E−03 2.3 24.8 Y C03_17B5 TALEN YC04_22C12 FokI 1.83E+04 4.83E−04 23.9 26.4 Y C04_22C12 TALEN Y D02_10B4FokI 5.86E+05 4.36E−03 2.6 7.4 Y D02_10B4 TALEN Y D04_23D1 FokI 2.63E+046.20E−04 18.6 23.6 Y D04_23D1 TALEN Y E01_2D7 FokI 7.45E+05 1.48E−02 0.819.9 Y E01_2D7 TALEN Y E02_10D7 FokI 5.15E+05 1.96E−02 0.6 38.1 YE02_10D7 TALEN Y G01_5E11 FokI 5.35E+05 2.01E−02 0.6 37.6 Y G01_5E11TALEN Y G02_11D2 FokI 1.59E+05 8.91E−04 13.0 5.6 Y G02_11D2 TALEN YG03_20F9 FokI 1.49E+06 4.65E−02 0.2 31.2 Y G03_20F9 TALEN Y G04_1A1 FokI5.00E+03 5.15E−04 22.4 103 Y G04_1A1 TALEN Y H01_6H4 FokI 2.97E+051.87E−03 6.2 6.3 Y H01_6H4 TALEN Y H02_13B2 FokI 1.06E+06 3.21E−02 0.430.3 Y H02_13B2 TALEN Y

Hybridoma fusion #2299 cells were obtained from a long immunization onday 94. Balb/c mouse #2299 with higher antibody titer was selected forfusion. Antibodies obtained from fusion #2299 resulted in over 68 ELISApositives screened on FokI and full-length TALEN protein. Theseantibodies were negative on the His tag antigen. Biacore® analysis offusion #2299 antibodies resulted in kD values ranging from sub nM to 100nM. Five out of the top 12 ELISA binders were positive in Western Blotanalysis with supernatant (FIG. 1 ). The Biacore® kinetics of exemplaryantibodies from fusion #2299 are shown in Table 3 below.

TABLE 3 Biacore ® kinetics of Fusion 2299 Binds FokI Ligand Analytek_(a) (1/Ms) k_(d) (1/s) t_(1/2) (min) K_(D) (nM) or TALEN (Y/N/A)D04_4C7 FokI 3.32E+05 1.26E−04 91.69 0.38 Y C09_9H1 FokI 5.72E+049.38E−05 123.16 1.64 Y A02_1G2 FokI 3.63E+04 6.12E−05 188.77 1.69 YC02_1G12 FokI 5.94E+04 1.16E−04 99.59 1.95 Y A04_4B10 FokI 1.02E+052.33E−04 49.58 2.28 Y G02_2E5 FokI 1.10E+05 2.73E−04 42.32 2.48 YE01_1D1 FokI 7.43E+04 1.91E−04 60.48 2.57 Y H08_8H12 FokI 2.39E+069.98E−03 1.16 4.18 Y B02_1G6 Fok 1.01E+05 4.36E−04 26.50 4.32 Y E02_2D12FokI 7.09E+04 3.29E−04 35.11 4.64 Y D01_1C10 FokI 7.47E+04 3.96E−0429.17 5.30 Y G09_2299.8C5 FokI 1.58E+05 1.21E−03 9.55 7.66 Y F08_8F3FokI 2.92E+04 2.59E−04 44.60 8.87 Y F09_2299.4D4.1 FokI 1.06E+041.39E−04 83.11 13.11 Y G07_7D2 FokI 9.06E+05 1.48E−02 0.78 16.34 YB08_7G8 FokI 9.16E+05 1.51E−02 0.77 16.48 Y H05_6A6 FokI 6.65E+031.17E−04 98.74 17.59 Y F07_7B11 FokI 6.60E+05 1.75E−02 0.66 26.52 YA01_1A4 FokI 7.66E+05 2.05E−02 0.56 26.76 Y A09_9D1 FokI 5.85E+052.74E−02 0.42 46.84 Y G04_4F11 FokI 6.99E+03 3.32E−04 34.80 47.50 YH01_1F12 FokI 4.30E+03 2.10E−04 55.01 48.84 Y E07_7A11 FokI 5.23E+052.60E−02 0.44 49.71 Y H02_2E8 FokI 9.54E+03 4.78E−04 24.17 50.10 YC03_3D1 FokI 5.27E+05 3.56E−02 0.32 67.55 Y E04_4D4 FokI 2.45E+05<8.55E−05  >135 <0.35 Y F04_4D12 FokI 2.42E+05 <8.55E−05  >135 <0.35 YC04_4C6 FokI 9.12E+04 <8.55E−05  >135 <0.94 Y A08_7F11 FokI 7.57E+04<8.55E−05  >135 <1.13 Y D08_8D2 FokI 7.23E+04 <8.55E−05  >135 <1.18 YF05_5G9 FokI 6.45E+04 <8.55E−05  >135 1.33 Y E05_5F5 FokI 5.84E+04<8.55E−05  >135 <1.46 Y B04_4B11 FokI 4.81E+04 <8.55E−05  >135 <1.78 YC08_8C7 FokI 4.81E+04 <8.55E−05  >135 <1.78 Y

Purified antibodies shown in Table 4 below were chosen for furtheranalysis and assay development.

TABLE 4 Summary of antibody panel Binds FokI or t_(1/2) TALEN WesternLigand Analyte k_(a) (1/Ms) k_(d) (1/s) (min) K_(D) (nM) (Y/N/A) ELISABlot 2299.2G3.C6 FokI 1.77E+04 <8.55E−05  >135 <4.83 Y + + 2299.5D4.C8FokI 1.12E+04 <8.55E−05  >135 <7.63 Y + + 2299.6B3.B8.B6 FokI 9.87E+03<8.55E−05  >135 <8.66 Y + + 2299.6G5.C7 FokI 5.27E+03 <8.55E−05  >135<16.2 Y + + 2299.7F11.B4 FokI 7.57E+04 <8.55E−05  >135 <1.13 Y + +2296.14H5.C10 FokI 1.39E+03 6.81E−04 16.96 489.93 Y +/− − 2296.17B5.A4FokI 8.33E+04 9.40E−04 12.29 11.28 Y + − 2296.23D1.B2 FokI 2.31E+036.31E−04 18.31 273.16 Y +/− −

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the present disclosure as described herein. The scope ofthe present disclosure is not intended to be limited to the abovedescription, but rather as set forth in the following claims.

What is claimed is:
 1. An anti-TALEN antibody comprising a variableheavy chain amino acid sequence that is at least 85% identical to SEQ IDNO: 3 and a variable light chain amino acid sequence that is at least85% identical to SEQ ID NO:
 7. 2. The anti-TALEN antibody of claim 1,wherein the antibody is humanized.
 3. The anti-TALEN antibody of claim 1or 2, wherein the antigen binding molecule is selected from the groupconsisting of an antibody, an scFv, a Fab, a Fab′, a Fv, a F(ab′)₂, adAb, a human antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a polyclonal antibody, a recombinant antibody, anIgE antibody, an IgD antibody, an IgM antibody, an IgG1 antibody, anIgG1 antibody having at least one mutation in the hinge region, an IgG2antibody an IgG2 antibody having at least one mutation in the hingeregion, an IgG3 antibody, an IgG3 antibody having at least one mutationin the hinge region, an IgG4 antibody, an IgG4 antibody having at leastone mutation in the hinge region, an antibody comprising at least onenon-naturally occurring amino acid, and any combination thereof.
 4. Theanti-TALEN antibody of any one of claims 1-3, wherein the antibodycomprises a variable heavy chain (HC) selected from the group consistingof SEQ ID NOs: 3-6.
 5. The anti-TALEN antibody of any one of thepreceding claims, comprising a heavy chain CDR1 selected from the groupconsisting of SEQ ID NOs: 12-14.
 6. The anti-TALEN antibody of any oneof the preceding claims, comprising a heavy chain CDR2 selected from thegroup consisting of SEQ ID NOs: 15-20.
 7. The anti-TALEN antibody of anyone of the preceding claims, comprising a heavy chain CDR3 comprisingSEQ ID NO:
 21. 8. The anti-TALEN antibody of any one of the precedingclaims, wherein the heavy chain comprises a heavy chain CDR1, a heavychain CDR2, and a heavy chain CDR3, each CDR comprising an amino acidsequence shown in Table 1c.
 9. An anti-TALEN antibody comprising a VHamino acid sequence that is at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or about 100% identical to a VH of an antigen binding molecule ofclaim
 4. 10. The anti-TALEN antibody any one of claims 1-9, wherein theisolated antibody comprises a light chain variable region (VL) sequenceselected from the group consisting of SEQ ID NOs: 7-11.
 11. Theanti-TALEN antibody of claim 10, comprising a light chain CDR1comprising SEQ ID NOs:
 22. 12. The anti-TALEN antibody of claim 10,comprising a light chain CDR2 selected from the group consisting of SEQID NO: 23-25.
 13. The anti-TALEN antibody of claim 10, comprising alight chain CDR3 selected from the group consisting of SEQ ID NOs:26-28.
 14. The anti-TALEN antibody of claim 10, wherein the light chaincomprises a light chain CDR1, a light chain CDR2 and a light chain CDR3,each CDR comprising an amino acid sequence in Table 1d.
 15. Ananti-TALEN antibody, comprising a VL amino acid sequence that is atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or about 100% identical toa VL of an antigen binding molecule of claim
 10. 16. An anti-TALENantibody, comprising (a) a VH CDR1 comprising the amino acid sequence ofSEQ ID NO:12; (b) a VH CDR2 comprising the amino acid sequence of SEQ IDNO: 15; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:21; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 22;(e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and(f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:
 26. 17.The anti-TALEN antibody of claim 16, comprising (a) a VH comprising theamino acid sequence of SEQ ID NO: 3; and (b) a VL comprising the aminoacid sequence of SEQ ID NO:
 7. 18. An anti-TALEN antibody, comprising(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (b) aVH CDR2 comprising the amino acid sequence of SEQ ID NO: 16; (c) a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 24; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 27. 19. The anti-TALENantibody of claim 18, comprising (a) a VH comprising the amino acidsequence of SEQ ID NO: 3; and (b) a VL comprising the amino acidsequence of SEQ ID NO:
 8. 20. An anti-TALEN antibody, comprising (a) aVH CDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16; (c) a VH CDR3comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 25; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 26. 21. The anti-TALENantibody of claim 20, comprising (a) a VH comprising the amino acidsequence of SEQ ID NO: 4; and (b) a VL comprising the amino acidsequence of SEQ ID NO:
 9. 22. An anti-TALEN antibody, comprising (a) aVH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (b) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 15; (c) a VH CDR3comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 24; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 28. 23. The anti-TALENantibody of claim 22, comprising (a) a VH comprising the amino acidsequence of SEQ ID NO: 5; and (b) a VL comprising the amino acidsequence of SEQ ID NO:
 10. 24. An anti-TALEN antibody, comprising (a) aVH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (b) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 17; (c) a VH CDR3comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 24; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 27. 25. The anti-TALENantibody of claim 24, comprising (a) a VH comprising the amino acidsequence of SEQ ID NO: 6; and (b) a VL comprising the amino acidsequence of SEQ ID NO:
 11. 26. An anti-TALEN antibody, comprising (a) aVH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (b) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 15; (c) a VH CDR3comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 24; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 27. 27. The anti-TALENantibody of claim 24, comprising (a) a VH comprising the amino acidsequence of SEQ ID NO: 3; and (b) a VL comprising the amino acidsequence of SEQ ID NO:
 8. 28. An anti-TALEN antibody, comprising (a) aVH CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (b) a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16; (c) a VH CDR3comprising the amino acid sequence of SEQ ID NO: 21; (d) a VL CDR1comprising the amino acid sequence of SEQ ID NO: 22; (e) a VL CDR2comprising the amino acid sequence of SEQ ID NO: 25; and (f) a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 26. 29. The anti-TALENantibody of claim 24, comprising (a) a VH comprising the amino acidsequence of SEQ ID NO: 4; and (b) a VL comprising the amino acidsequence of SEQ ID NO:
 9. 30. The anti-TALEN antibody of any of claims1-29, wherein the isolated antibody further comprises a detectablelabel.
 31. The anti-TALEN antibody of claim 30, wherein the detectablelabel is selected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten.
 32. The anti-TALEN antibody of claim31, wherein the fluorescent label is selected from the group consistingof an Atto dye, an Alexafluor dye, quantum dots, Hydroxycoumarin,Aminocouramin, Methoxycourmarin, Cascade Blue, Pacific Blue, PacificOrange, Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates,PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein,BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine,Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC), APC-Cy7conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP,Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, KusabiraOrange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP,DsRed monomer, DsRed2 (“RFP”), mStrawberry, TurboFP602, AsRed2, mRFP1,J-Red, R-phycoerythrin (RPE), B-phycoeryhring (BPE), mCherry, HcRed1,Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635),TurboFP635, mPlum, and mRaspberry.
 33. The anti-TALEN antibody of claim32, wherein the fluorescent label is R-Phycoerythrin (PE) orAllophycocyanin (APC).
 34. A composition comprising the anti-TALENantibody of any one of claims 1-33 and a pharmaceutically acceptablecarrier or vehicle.
 35. A polynucleotide encoding the heavy chain of ananti-TALEN antibody of any one of claims 1-29.
 36. A polynucleotideencoding the light chain of an anti-TALEN antibody of any one of claims1-29.
 37. A vector comprising the polynucleotide of claim 35 or claim36.
 38. A cell comprising one or both of the vectors of claim
 37. 39.The cell of claim 38, wherein the cell comprises a cell selected fromthe group consisting of a CHO cell, a Sp2/0 cell, a rabbit cell and anE. coli cell.
 40. A method of making an anti-TALEN antibody of claims1-29 comprising incubating the cell of claim 39 under suitableconditions.
 41. A method of determining the presence or absence of aTALEN protein in a sample, comprising contacting the sample with ananti-TALEN antibody conjugated to a detectable label and determining thepresence or absence of the TALEN protein in the sample.
 42. The methodof claim 41, wherein the anti-TALEN antibody comprises an antibody ofany one of claims 1-33 or a humanized form thereof.
 43. The method ofclaim 41 or 42, wherein the sample is a tissue sample, a blood sample, aformalin-fixed sample, a tissue grown ex vivo or cell culture media.